Claudin-6-specific immunoreceptors and t cell epitopes

ABSTRACT

The present invention provides Claudin-6-specific immunoreceptors (T cell receptors and artificial T cell receptors (chimeric antigen receptors; CARs)) and T cell epitopes which are useful for immunotherapy.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application continuation of U.S. patent application Ser. No.16/444,510, filed on Jun. 18, 2019, which is a divisional application ofU.S. patent application Ser. No. 15/124,640, filed on Sep. 8, 2016,which was a U.S. National Stage Application under 35 U.S.C. § 371 ofInternational Patent Application No. PCT/EP2015/056899, filed Mar. 30,2015, which claims the benefit of priority of European PatentApplication numbers PCT/EP2014/000868, filed Apr. 1, 2014, andPCT/EP2014/072864, filed Oct. 24, 2014, all of which are incorporated byreference in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

A sequence listing, filed as the ASCII text file “Sequence List” havinga file size of 87 kilobytes, is incorporated herein by reference in itsentirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the provision of Claudin-6-specificimmunoreceptors (T cell receptors and artificial T cell receptors(chimeric antigen receptors; CARs)) and T cell epitopes which are usefulfor immunotherapy.

BACKGROUND OF THE INVENTION

The evolution of the immune system resulted in vertebrates in a highlyeffective network based on two types of defense: the innate and theadoptive immunity.

In contrast to the evolutionary ancient innate immune system that relieson invariant receptors recognizing common molecular patterns associatedwith pathogens, the adoptive immunity is based on highly specificantigen receptors on B cells (B lymphocytes) and T cells (T lymphocytes)and clonal selection.

While B cells raise humoral immune responses by secretion of antibodies,T cells mediate cellular immune responses leading to destruction ofrecognized cells.

T cells play a central role in cell-mediated immunity in humans andanimals. The recognition and binding of a particular antigen is mediatedby the T cell receptors (TCRs) expressed on the surface of T cells.

The T cell receptor (TCR) of a T cell is able to interact withimmunogenic peptides (epitopes) bound to major histocompatibilitycomplex (MHC) molecules and presented on the surface of target cells.Specific binding of the TCR triggers a signal cascade inside the T cellleading to proliferation and differentiation into a maturated effector Tcell. To be able to target a vast variety of antigens, the T cellreceptors need to have a great diversity.

This diversity is obtained by genetic rearrangement of differentdiscontinuous segments of genes which code for the different structuralregions of TCRs. TCRs are composed of one α-chain and one β-chain or ofone γ-chain and one δ-chain. The TCR α/β chains are composed of anN-terminal highly polymorphic variable region involved in antigenrecognition and an invariant constant region. On the genetic level,these chains are separated into several regions, a variable (V) region,a diversity (D) region (only β- and δ-chain), a joining (J) region and aconstant (C) region. The human β-chain genes contain over 60 variable(V), 2 diversity (D), over 10 joining (J) segments, and 2 constantregion segments (C). The human α-chain genes contain over 50 V segments,and over 60 J segments but no D segments, as well as one C segment. Themurine β-chain genes contain over 30 variable (V), 2 diversity (D), over10 joining (J) segments, and 2 constant region segments (C). The murineα-chain genes contain almost 100 V segments, 60 J segments, no Dsegments, but one C segment. During the differentiation of T cells,specific T cell receptor genes are created by rearranging one V, one D(only β- and δ-chain), one J and one C region gene. The diversity of theTCRs is further amplified by imprecise V-(D)-J rearrangement whereinrandom nucleotides are introduced and/or deleted at the recombinationsites. Since the rearrangement of the TCR gene loci occurs in the genomeduring maturation of T cells, each mature T cell only expresses onespecific α/β TCR or γ/δ TCR.

MHC and antigen binding is mediated by the complementary determiningregions 1, 2 and 3 (CDR1, CDR2, CDR3) of the TCR. The CDR3 of theβ-chain which is most critical for antigen recognition and binding isencoded by the V-D-J junction of the rearranged TCR β-chain gene. TheTCR is a part of a complex signaling machinery, which includes theheterodimeric complex of the TCR α- and β-chains, the co-receptor CD4 orCD8 and the CD3 signal transduction module (FIG. 1). While the CD3chains transfer the activation signal inside the cell, the TCR α/βheterodimer is solely responsible for antigen recognition. Thus, thetransfer of the TCR α/β chains offers the opportunity to redirect Tcells towards any antigen of interest.

Immunotherapy

Antigen-specific immunotherapy aims to enhance or induce specific immuneresponses in patients to control infectious or malignant diseases. Theidentification of a growing number of pathogen- and tumor-associatedantigens (TAA) led to a broad collection of suitable targets forimmunotherapy. Cells presenting immunogenic peptides (epitopes) derivedfrom these antigens can be specifically targeted by either active orpassive immunization strategies.

Active immunization tends to induce and expand antigen-specific T cellsin the patient, which are able to specifically recognize and killdiseased cells. In contrast passive immunization relies on the adoptivetransfer of T cells, which were expanded and optional geneticallyengineered in vitro (adoptive T cell therapy).

Vaccination

Tumor vaccines aim to induce endogenous tumor-specific immune responsesby active immunization. Different antigen formats can be used for tumorvaccination including whole cancer cells, proteins, peptides orimmunizing vectors such as RNA, DNA or viral vectors that can be appliedeither directly in vivo or in vitro by pulsing of DCs following transferinto the patient.

The number of clinical studies where therapy-induced immune responsescan be identified is steadily increasing due to improvements ofimmunization strategies and methods for detection of antigen-specificimmune responses (Connerotte, T. et al. (2008). Cancer Res. 68,3931-3940; Schmitt, M. et al. (2008) Blood 111, 1357-1365; Speiser, D.E. et al. (2008) Proc. Natl. Acad. Sci. U.S.A 105, 3849-3854; Adams, S.et al. (2008) J. Immunol. 181, 776-784).

However, in most cases detected immune responses cannot systemically becorrelated with clinical outcomes (Curigliano, G. et al. (2006) Ann.Oncol. 17, 750-762; Rosenberg, S. A. et al. (2004) Nat. Med. 10,909-915).

The exact definition of peptide epitopes derived from tumor antigens maytherefore contribute to improve specificity and efficiency ofvaccination strategies as well as methods for immunomonitoring.

Adoptive Cell Transfer (ACT)

ACT based immunotherapy can be broadly defined as a form of passiveimmunization with previously sensitized T cells that are transferred tonon-immune recipients or to the autologous host after ex vivo expansionfrom low precursor frequencies to clinically relevant cell numbers. Celltypes that have been used for ACT experiments are lymphokine-activatedkiller (LAK) cells (Mule, J. J. et al. (1984) Science 225, 1487-1489;Rosenberg, S. A. et al. (1985) N. Engl. J. Med. 313, 1485-1492),tumor-infiltrating lymphocytes (TILs) (Rosenberg, S. A. et al. (1994) J.Natl. Cancer Inst. 86, 1159-1166), donor lymphocytes after hematopoieticstem cell transplantation (HSCT) as well as tumor-specific T cell linesor clones (Dudley, M. E. et al. (2001) J. Immunother. 24, 363-373; Yee,C. et al. (2002) Proc. Natl. Acad. Sci. U.S.A 99, 16168-16173). AdoptiveT cell transfer was shown to have therapeutic activity against humanviral infections such as CMV. While CMV infection and reactivation ofendogenous latent viruses is controlled by the immune system in healthyindividuals, it results in significant morbidity and mortality in immunecompromised individuals such as transplant recipients or AIDS patients.

Riddell and co-workers demonstrated the reconstitution of viral immunityby adoptive T cell therapy in immune suppressed patients after transferof CD8+ CMV-specific T cell clones derived from HLA-matchedCMV-seropositive transplant donors (Riddell, S. R. (1992) Science 257,238-241).

As an alternative approach polyclonal donor-derived CMV- or EBV-specificT cell populations were transferred to transplant recipients resultingin increased persistence of transferred T cells (Rooney, C. M. et al.(1998) Blood 92, 1549-1555; Peggs, K. S. et al. (2003) Lancet 362,1375-1377).

For adoptive immunotherapy of melanoma Rosenberg and co-workersestablished an ACT approach relying on the infusion of in vitro expandedautologous tumor-infiltrating lymphocytes (TILs) isolated from excisedtumors in combination with a non-myeloablative lymphodepletingchemotherapy and high-dose IL2. A recently published clinical studyresulted in an objective response rate of ˜50% of treated patientssuffering from metastatic melanoma (Dudley, M. E. et al. (2005) J. Clin.Oncol. 23: 2346-2357).

However, patients must fulfill several premises to be eligible for ACTimmunotherapy. They must have resectable tumors. The tumors mustgenerate viable TILs under cell culture conditions. The TILs must bereactive against tumor antigens, and must expand in vitro to sufficientnumbers. Especially in other cancers than melanoma, it is difficult toobtain such tumor-reactive TILs. Furthermore, repeated in vitrostimulation and clonal expansion of normal human T lymphocytes resultsin progressive decrease in telomerase activity and shortening oftelomeres resulting in replicative senescence and decreased potentialfor persistence of transferred T cells (Shen, X. et al. (2007) J.Immunother. 30: 123-129).

ACT Using Gene-Engineered T Cells

An approach overcoming the limitations of ACT is the adoptive transferof autologous T cells reprogrammed to express a tumor-reactiveimmunoreceptor of defined specificity during short-time ex vivo culturefollowed by reinfusion into the patient (Kershaw M. H. et al. (2013)Nature Reviews Cancer 13 (8):525-41). This strategy makes ACT applicableto a variety of common malignancies even if tumor-reactive T cells areabsent in the patient. Since the antigenic specificity of T cells isrested entirely on the heterodimeric complex of the TCR α- and β-chain,the transfer of cloned TCR genes into T cells offers the potential toredirect them towards any antigen of interest. Therefore, TCR genetherapy provides an attractive strategy to develop antigen-specificimmunotherapy with autologous lymphocytes as treatment option. Majoradvantages of TCR gene transfer are the creation of therapeuticquantities of antigen-specific T cells within a few days and thepossibility to introduce specificities that are not present in theendogenous TCR repertoire of the patient.

Several groups demonstrated, that TCR gene transfer is an attractivestrategy to redirect antigen-specificity of primary T cells (Morgan, R.A. et al. (2003) J. Immunol. 171, 3287-3295; Cooper, L. J. et al. (2000)J. Virol. 74, 8207-8212; Fujio, K. et al. (2000) J. Immunol. 165,528-532; Kessels, H. W. et al. (2001) Nat. Immunol. 2, 957-961; Dembic,Z. et al. (1986) Nature 320, 232-238).

Feasibility of TCR gene therapy in humans was recently demonstrated inclinical trials for the treatment of malignant melanoma by Rosenberg andhis group. The adoptive transfer of autologous lymphocytes retrovirallytransduced with melanoma/melanocyte antigen-specific TCRs resulted incancer regression in up to 30% of treated melanoma patients (Morgan, R.A. et al. (2006) Science 314, 126-129; Johnson, L. A. et al. (2009)Blood 114, 535-546).

Chimeric Antigen Receptors

Chimeric antigen receptors (CARs) are engineered receptors that combinea single chain variable fragment (scFv) of a monoclonal antibody with anintracellular part consisting of one or more signaling domains for Tcell activation. CARs recognize native antigens in a non-MHC-restrictedmanner and can therefore be used in all individuals no matter what theirHLA type is and they are functional in CD4+ as well as CD8+ T cells.

A multitude of CARs has been reported over the past decade, targeting apanel of different cell surface tumor antigens. Their biologic functionswere dramatically improved by incorporation of a costimulatory domainresulting in tripartite receptors (scFv, CD28, CD3ζ), termed 2ndgeneration CARs. CARs of the 3rd generation encompass additional domainsof costimulatory molecules such as OX40 and 4-1BB to enhance theproliferative capacity and persistence of modified T-cells (FIG. 2).

Target Structures for Antigen-Specific Immunotherapy

The discovery of multiple tumor-associated antigens (TAAs) has providedthe basis for antigen-specific immunotherapy concepts (Novellino, L. etal. (2005) Cancer Immunol. Immunother. 54, 187-207). TAAs are unusualproteins expressed on tumor cells due to their genetic instability,which have no or limited expression in normal cells. These TAAs can leadto specific recognition of malignant cells by the immune system.

Molecular cloning of TAAs by screening of tumor-derived cDNA expressionlibraries using autologous tumor-specific T cells (van der Bruggen, P.et al. (1991) Science 254, 1643-1647) or circulating antibodies (Sahin,U. et al. (1995) Proc. Natl. Acad. Sci. U.S.A 92, 11810-11813), reverseimmunology approaches, biochemical methods (Hunt, D. F. et al. (1992)Science 256, 1817-1820), gene expression analyses or in silico cloningstrategies (Helftenbein, G. et al. (2008) Gene 414, 76-84) led to asignificant number of target candidates for immunotherapeuticstrategies. TAAs fall in several categories, including differentiationantigens, overexpressed antigens, tumor-specific splice variants,mutated gene products, viral and cancer testis antigens (CTAs). Thecancer testis family is a very promising category of TAAs as theirexpression is restricted to the testis and a multitude of differenttumor entities (Scanlan, M. J. et al. (2002) Immunol. Rev. 188, 22-32).Until now more than 50 CT genes have been described (Scanlan, M. J. etal. (2004) Cancer Immun 4, 1) and some of them have been addressed inclinical studies (Adams, S. et al. (2008) J. Immunol. 181, 776-784;Atanackovic, D. et al. (2004) J. Immunol. 172, 3289-3296; Chen, Q. etal. (2004) Proc. Natl. Acad. Sci. U.S.A 101, 9363-9368; Connerotte, T.et al. (2008). Cancer Res. 68, 3931-3940; Davis, I. D. et al. (2004)Proc. Natl. Acad. Sci. U.S.A 101, 10697-10702; Jager, E. (2000) Proc.Natl. Acad. Sci. U.S.A 97, 12198-12203; Marchand, M. et al. (1999) Int.J. Cancer 80, 219-230; Schuler-Thurner, B. et al. (2000) J. Immunol.165, 3492-3496).

In spite of the growing number of attractive target structures forimmunotherapeutic approaches specific T cell clones or lines of definedHLA restriction do only exist for a few of them (Chaux, P. et al. (1999)J. Immunol. 163, 2928-2936; Zhang, Y. et al. (2002) Tissue Antigens 60,365-371; Zhao, Y. et al. (2005) J. Immunol. 174, 4415-4423).

Claudins are integral membrane proteins located within the tightjunctions of epithelia and endothelia. Claudins are predicted to havefour transmembrane segments with two extracellular loops, and N- andC-termini located in the cytoplasm. The Claudin (CLDN) family oftransmembrane proteins plays a critical role in the maintenance ofepithelial and endothelial tight junctions and might also play a role inthe maintenance of the cytoskeleton and in cell signalling.

Claudin-6 (CLDN6) is an oncofetal gene expressed in murine and humanstem cells as well as embryoid bodies committed to the epithelia cellfate (Turksen, K. et al. (2001) Dev Dyn 222, 292-300; Anderson W J. etal. (2008) Dev Dyn 237, 504-12; Turksen K. et al. (2002) Development,129, 1775-84; Assou S. et al. (2007) Stem Cells 25, 961-73). As atumor-associated antigen it can be classified as a differentiationantigen due to its expression during early stage of epidermalmorphogenesis where it is crucial for epidermal differentiation andbarrier formation. Additionally expression was observed in epithelialtissues or neonatal normal epithelial tissue of tongue, skin, stomachand breast (Abuazza G. et al. (2006), Am J Physiol Renal Physiol 291,1132-1141; Troy T. C. et al. (2007), Molecular Biotechnology 36, 166-74;Zhao L. et al. (2008), Am J Physiol Regul Integr Comp Physiol 294,1856-1862). Besides that, own data also reveal low or very lowexpression of CLDN6 in human placenta, urinary bladder, endometrium,prostate and the peripheral nerve and frequent overexpression of CLDN6in different cancers. CLDN6 has been demonstrated to be overexpressed intumors, including pediatric brain tumors, gastric adenocarcinomas andgerm cell tumors as well as visceral carcinomas such as ovariancarcinomas (FIG. 4). It has also been demonstrated that overexpressionof CLDN6 in gastric cancer cells results in increased invasiveness,migration and proliferation suggesting that CLDN6 is a marker for poorprognosis and may play a potential role in maintaining the malignantphenotype. In addition, it has been shown that CLDN6 functions as cancersuppressor via inhibition of cell proliferation and induction ofapoptosis in breast cancer cell lines.

The frequent overexpression of CLDN6 on tumors qualifies this moleculeas a highly attractive target for development of therapeutics directedagainst CLDN6 such as vaccine therapeutics and therapeutic antibodies.However, hitherto no HLA-A*2-restricted CLDN6 T cell epitopes and T cellreceptors targeting CLDN6 have been described and it is unknown whetherCLDN6 expressing cancer cells can be targeted in vivo by immunotherapiesinvolving T cells using active or passive immunization approaches.

DESCRIPTION OF INVENTION Summary of the Invention

The present invention relates to T cell receptors and artificial T cellreceptors specific for the tumor-associated antigen CLDN6, in particularwhen present on the surface of a cell such as a diseased cell orpresented on the surface of a cell such as a diseased cell or anantigen-presenting cell, as well as peptides comprising epitopesrecognized by these T cell receptors, i.e. CLDN6-T cell epitopes.

By adoptive transfer of T cells engineered to express such T cellreceptor or artificial T cell receptor CLDN6 expressing cancer cells canbe specifically targeted thereby leading to selective destruction ofcancer cells. Furthermore, the T cell epitopes provided according to theinvention are useful for designing vaccines against CLDN6-expressingcancers.

In one aspect, the invention relates to a peptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 3, 4 and5 or a variant of said amino acid sequence. In one embodiment thepeptide is 100 or less, 50 or less, 20 or less, or 10 or less aminoacids long. In one embodiment, the peptide can be processed to produce apeptide consisting of the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 3, 4 and 5 or a variant of said amino acidsequence. In one embodiment, the peptide consists of the amino acidsequence selected from the group consisting of SEQ ID NOs: 3, 4 and 5 ora variant of said amino acid sequence.

In one embodiment, the peptide is a MHC class I or class II presentedpeptide, preferably a MHC class I presented peptide, or, if presentwithin cells, can be processed to produce a procession product thereofwhich is a MHC class I or class II presented peptide, preferably a MHCclass I presented peptide. Preferably, said MHC class I or class IIpresented peptide has a sequence substantially corresponding to thegiven amino acid sequence, i.e. an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 3, 4 and 5 or a variant of said aminoacid sequence. Preferably, a peptide according to the invention iscapable of stimulating a cellular response against a disease involvingcells characterized by presentation of CLDN6 with class I MHC.

In further aspects, the invention relates to a nucleic acid comprising anucleotide sequence encoding the peptide of the invention and a cellcomprising the nucleic acid. The nucleic acid may be a recombinantnucleic acid. The nucleic acid may be present in a plasmid or anexpression vector and may be functionally linked to a promoter. In oneembodiment, the nucleic acid is RNA. Preferably, the cell expresses thepeptide. The cell may be a recombinant cell and may secrete the encodedpeptide or a procession product thereof, may express it on the surfaceand preferably may additionally express an MHC molecule which binds tosaid peptide or a procession product thereof and preferably presentssaid peptide or a procession product thereof on the cell surface. In oneembodiment, the cell expresses the MHC molecule endogenously. In afurther embodiment, the cell expresses the MHC molecule and/or thepeptide in a recombinant manner. The cell is preferablynonproliferative. In a preferred embodiment, the cell is anantigen-presenting cell, in particular a dendritic cell, a monocyte or amacrophage.

In a further aspect, the invention relates to a cell that presents thepeptide of the invention or a procession product thereof, wherein theprocession product preferably is a peptide having the given amino acidsequence, i.e. an amino acid sequence selected from the group consistingof SEQ ID NOs: 3, 4 and 5 or a variant of said amino acid sequence. Inone embodiment, said cell is a cell comprising a nucleic acid comprisinga nucleotide sequence encoding the peptide of the invention. Preferablysaid cell expresses said nucleic acid so as to produce said peptide.Optionally said cell processes said peptide so as to produce a peptideconsisting of an amino acid sequence selected from the group consistingof SEQ ID NOs: 3, 4 and 5 or a variant of said amino acid sequence. Thecell may present the peptide or a procession product thereof by MHCmolecules on its surface. In one embodiment, the cell endogenouslyexpresses an MHC molecule. In a further embodiment, the cellrecombinantly expresses an MHC molecule. In one embodiment, the MHCmolecules of the cell are loaded (pulsed) with the peptide by additionof the peptide to the cell. The cell may recombinantly express thepeptide and present said peptide or a procession product thereof on thecell surface. The cell is preferably nonproliferative. In a preferredembodiment, the cell is an antigen-presenting cell such as a dendriticcell, a monocyte or a macrophage.

In a further aspect, the invention relates to an immunoreactive cellwhich is reactive with a peptide of the invention, in particular whenpresented on the surface of a cell such as a diseased cell. Theimmunoreactive cell may be a cell that has been sensitized in vitro torecognize the peptide. The immunoreactive cell may be a T cell,preferably a cytotoxic T cell. Preferably, the immunoreactive cell bindsto a sequence substantially corresponding to the given amino acidsequence, i.e. an amino acid sequence selected from the group consistingof SEQ ID NOs: 3, 4 and 5 or a variant of said amino acid sequence, inparticular when bound to MHC such as MHC on the surface of a cell suchas a diseased cell.

In a further aspect, the invention relates to a binding agent whichbinds to a peptide of the invention, optionally in a complex with an MHCmolecule.

In a further aspect, the invention relates to a T cell receptor whichbinds to a peptide of the invention, optionally in a complex with an MHCmolecule, and preferably is reactive with said peptide, or a polypeptidechain of said T cell receptor. In one embodiment, the polypeptide chainof said T cell receptor is a T cell receptor α-chain or T cell receptorβ-chain.

In a further aspect, the invention relates to a T cell receptor α-chainor a T cell receptor comprising said T cell receptor α-chain, whereinsaid T cell receptor α-chain is selected from the group consisting of:

(i) a T cell receptor α-chain comprising at least one, preferably two,more preferably all three of the CDR sequences of a T cell receptorα-chain selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26 and 28 or a variant thereof and

(ii) a T cell receptor α-chain comprising a T cell receptor α-chainsequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26 and 28 or a fragment thereof, or a variant ofsaid sequence or fragment.

In one embodiment, said SEQ ID NOs: are selected from the groupconsisting of SEQ ID NOs: 6, 8, 10, 12, 14 and 16 and said T cellreceptor is reactive with a peptide comprising the amino acid sequenceof SEQ ID NO: 3 or a variant of said amino acid sequence.

In one embodiment, said SEQ ID NOs: are selected from the groupconsisting of SEQ ID NOs: 18, 20, 22, 24 and 26 and said T cell receptoris reactive with a peptide comprising the amino acid sequence of SEQ IDNO: 4 or a variant of said amino acid sequence.

In one embodiment, said SEQ ID NO: is SEQ ID NO: 28 and said T cellreceptor is reactive with a peptide comprising the amino acid sequenceof SEQ ID NO: 5 or a variant of said amino acid sequence.

In a further aspect, the invention relates to a T cell receptor β-chainor a T cell receptor comprising said T cell receptor β-chain,

wherein said T cell receptor β-chain is selected from the groupconsisting of:

(i) a T cell receptor β-chain comprising at least one, preferably two,more preferably all three of the CDR sequences of a T cell receptorβ-chain selected from the group consisting of SEQ ID NOs: 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27 and 29 or a variant thereof

and

(ii) a T cell receptor β-chain comprising a T cell receptor β-chainsequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27 and 29 or a fragment thereof, or a variant ofsaid sequence or fragment.

In one embodiment, said SEQ ID NOs: are selected from the groupconsisting of SEQ ID NOs: 7, 9, 11, 13, 15 and 17 and said T cellreceptor is reactive with a peptide comprising the amino acid sequenceof SEQ ID NO: 3 or a variant of said amino acid sequence.

In one embodiment, said SEQ ID NOs: are selected from the groupconsisting of SEQ ID NOs: 19, 21, 23, 25 and 27 and said T cell receptoris reactive with a peptide comprising the amino acid sequence of SEQ IDNO: 4 or a variant of said amino acid sequence.

In one embodiment, said SEQ ID NO: is SEQ ID NO: 29 and said T cellreceptor is reactive with a peptide comprising the amino acid sequenceof SEQ ID NO: 5 or a variant of said amino acid sequence.

In a further aspect, the invention relates to a T cell receptor selectedfrom the group consisting of:

(I) a T cell receptor comprising:

(i) a T cell receptor α-chain comprising at least one, preferably two,more preferably all three of the CDR sequences of the T cell receptorα-chain of SEQ ID NO: x or a variant thereof, and

(ii) a T cell receptor β-chain comprising at least one, preferably two,more preferably all three of the CDR sequences of a T cell receptorβ-chain of SEQ ID NO: x+1 or a variant thereof; wherein x selected fromthe group consisting of 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28

and

(II) a T cell receptor comprising:

(i) a T cell receptor α-chain comprising the T cell receptor α-chainsequence of SEQ ID NO: x or a fragment thereof, or a variant of saidsequence or fragment, and

(ii) a T cell receptor β-chain comprising the T cell receptor β-chainsequence of SEQ ID NO: x+1 or a fragment thereof, or a variant of saidsequence or fragment;

wherein x selected from the group consisting of 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26 and 28.

In one embodiment, said x is selected from the group consisting of 6, 8,10, 12, 14 and 16 and said T cell receptor is reactive with a peptidecomprising the amino acid sequence of SEQ ID NO: 3 or a variant of saidamino acid sequence.

In one embodiment, said x is selected from the group consisting of 18,20, 22, 24 and 26 and said T cell receptor is reactive with a peptidecomprising the amino acid sequence of SEQ ID NO: 4 or a variant of saidamino acid sequence.

In one embodiment, said x is 28 and said T cell receptor is reactivewith a peptide comprising the amino acid sequence of SEQ ID NO: 5 or avariant of said amino acid sequence.

In one embodiment, binding of said T cell receptor when expressed by Tcells and/or present on T cells to CLDN6-peptide epitopes as describedabove presented on cells such as cancer cells results in proliferationand/or activation of said T cells, wherein said activated T cellspreferably release cytotoxic factors, e.g. performs and granzymes, andinitiate cytolysis and/or apoptosis of cancer cells.

In a further aspect, the invention relates to an artificial T cellreceptor which binds to claudin-6 (CLDN6). In one embodiment, binding isa specific binding.

In one embodiment, said CLDN6 is expressed in a cancer cell. In oneembodiment said CLDN6 is expressed on the surface of a cancer cell. Inone embodiment said artificial T cell receptor binds to an extracellulardomain or to an epitope in an extracellular domain of CLDN6. In oneembodiment said artificial T cell receptor binds to native epitopes ofCLDN6 present on the surface of living cells. In one embodiment saidartificial T cell receptor binds to the first extracellular loop ofCLDN6. In one embodiment, binding of said artificial T cell receptorwhen expressed by T cells and/or present on T cells to CLDN6 present oncells such as cancer cells results in proliferation and/or activation ofsaid T cells, wherein said activated T cells preferably releasecytotoxic factors, e.g. perforins and granzymes, and initiate cytolysisand/or apoptosis of cancer cells.

In one embodiment, the artificial T cell receptor of the inventioncomprises a binding domain for CLDN6. In one embodiment, the bindingdomain for CLDN6 is comprised by an exodomain of said artificial T cellreceptor. In one embodiment, the binding domain for CLDN6 comprises asingle-chain variable fragment (scFv) of a CLDN6 antibody. In oneembodiment, the binding domain for CLDN6 comprises a variable region ofa heavy chain of an immunoglobulin (VH) with a specificity for CLDN6(VH(CLDN6)) and a variable region of a light chain of an immunoglobulin(VL) with a specificity for CLDN6 (VL(CLDN6)). In one embodiment, saidheavy chain variable region (VH) and the corresponding light chainvariable region (VL) are connected via a peptide linker, preferably apeptide linker comprising the amino acid sequence (GGGGS)3. In oneembodiment, the binding domain for CLDN6 comprises a VH(CLDN6)comprising an amino acid sequence represented by SEQ ID NO: 32 or afragment thereof, or a variant of said amino acid sequence or fragment.In one embodiment, the binding domain for CLDN6 comprises a VL(CLDN6)comprising an amino acid sequence represented by SEQ ID NO: 33, 38 or 39or a fragment thereof, or a variant of said amino acid sequence orfragment. In one embodiment, the binding domain for CLDN6 comprises aVH(CLDN6) comprising an amino acid sequence represented by SEQ ID NO: 32or a fragment thereof, or a variant of said amino acid sequence orfragment and a VL(CLDN6) comprising an amino acid sequence representedby SEQ ID NO: 39 or a fragment thereof, or a variant of said amino acidsequence or fragment. In one embodiment, the binding domain for CLDN6comprises an amino acid sequence represented by SEQ ID NO: 40 or afragment thereof, or a variant of said amino acid sequence or fragment.

In one embodiment, the artificial T cell receptor of the inventioncomprises a transmembrane domain. In one embodiment, the transmembranedomain is a hydrophobic alpha helix that spans the membrane. In oneembodiment, the transmembrane domain comprises the CD28 transmembranedomain or a fragment thereof.

In one embodiment, the artificial T cell receptor of the inventioncomprises a T cell signaling domain. In one embodiment, the T cellsignaling domain is located intracellularly. In one embodiment, the Tcell signaling domain comprises CD3-zeta, preferably the endodomain ofCD3-zeta, optionally in combination with CD28. In one embodiment, the Tcell signaling domain comprises the sequence according to SEQ ID NO: 45or a fragment thereof, or a variant of said sequence or fragment.

In one embodiment, the artificial T cell receptor of the inventioncomprises a signal peptide which directs the nascent protein into theendoplasmic reticulum. In one embodiment, the signal peptide precedesthe binding domain for CLDN6. In one embodiment, the signal peptidecomprises the sequence according to SEQ ID NO: 42 or a fragment thereof,or a variant of said sequence or fragment.

In one embodiment, the artificial T cell receptor of the inventioncomprises a spacer region which links the binding domain for CLDN6 tothe transmembrane domain. In one embodiment, the spacer region allowsthe binding domain for CLDN6 to orient in different directions tofacilitate CLDN6 recognition. In one embodiment, the spacer regioncomprises the hinge region from IgG1. In one embodiment, the spacerregion comprises the sequence according to SEQ ID NO: 43 or a fragmentthereof, or a variant of said sequence or fragment.

In one embodiment, the artificial T cell receptor of the inventioncomprises the structure:

NH2-signal peptide-binding domain for CLDN6-spacer region-transmembranedomain-T cell signaling domain-COOH.

In one embodiment, the artificial T cell receptor of the inventioncomprises the amino acid sequence according to SEQ ID NO: 46 or afragment thereof, or a variant of said amino acid sequence or fragment.

The above T cell receptors and artificial T cell receptors arepreferably specific for the tumor-associated antigen CLDN6, inparticular when present on the surface of a cell such as a diseased cellor when presented on the surface of a cell such as a diseased cell or anantigen-presenting cell.

The T cell receptors and artificial T cell receptors of the inventionmay be expressed by and/or present on the surface of cells such as Tcells.

In a further aspect, the invention relates to a nucleic acid comprisinga nucleotide sequence encoding the T cell receptor chain or T cellreceptor of the invention or encoding the artificial T cell receptor ofthe invention. In one embodiment, the nucleic acid is a recombinantnucleic acid. In one embodiment, the nucleic acid is in the form of avector or in the form of RNA.

In a further aspect, the invention relates to a cell comprising the Tcell receptor chain or T cell receptor of the invention or theartificial T cell receptor of the invention and/or comprising a nucleicacid comprising a nucleotide sequence encoding the T cell receptor chainor T cell receptor of the invention or encoding the artificial T cellreceptor of the invention. In one embodiment, said nucleic acid is RNA,preferably in vitro transcribed RNA. The cell may be a cell expressingthe T cell receptor chain or T cell receptor of the invention or theartificial T cell receptor of the invention and/or may have the T cellreceptor chain or T cell receptor of the invention or the artificial Tcell receptor of the invention on its cell surface. In one embodiment,said cell is a cell which is useful for adoptive cell transfer. The cellmay be an effector or stem cell, preferably an immunoreactive cell. Theimmunoreactive cell may be a T cell, preferably a cytotoxic T cell. Inone embodiment, the immunoreactive cell is reactive with thetumor-associated antigen CLDN6. In one embodiment, said CLDN6 is presenton the surface of a cell such as a diseased cell. In one embodiment,said CLDN6 is presented on the surface of a cell such as a diseased cellor an antigen-presenting cell, and the immunoreactive cell is reactivewith a peptide of the invention, in particular when presented in thecontext of MHC, and preferably binds to a sequence substantiallycorresponding to the given amino acid sequence, i.e. an amino acidsequence selected from the group consisting of SEQ ID NOs: 3, 4 and 5 ora variant of said amino acid sequence. In one embodiment, said celllacks surface expression of an endogenous TCR or is specific for aCLDN6-unrelated antigen.

In one embodiment, cells of the invention prior to use in adoptive celltransfer are subjected to an antigen-specific expansion and rechallenge,wherein the antigen-specific expansion and rechallenge may be effectedby exposing the cells to preferably autologous antigen presenting cellspresenting CLDN6 or a peptide fragment thereof.

In a further aspect, the invention relates to a method of producing animmunoreactive cell comprising the step of transducing a T cell with anucleic acid comprising a nucleotide sequence encoding the T cellreceptor chain or T cell receptor of the invention or encoding theartificial T cell receptor of the invention.

Furthermore, the present invention generally embraces the treatment ofdiseases by targeting diseased cells such as cancer cells, in particularcancer cells expressing CLDN6. The methods provide for the selectiveeradication of cells that express on their surface and/or present thetumor-associated antigen CLDN6, thereby minimizing adverse effects tonormal cells not expressing and/or presenting CLDN6. Thus, preferreddiseases for a therapy are those in which CLDN6 is expressed andoptionally presented such as cancer diseases, in particular thosedescribed herein.

When a peptide of the invention, a nucleic acid comprising a nucleotidesequence encoding the peptide of the invention or a cell of theinvention comprising said nucleic acid is administered, the treatmentpreferably involves an active immunization. Preferably, CLDN6-specific Tcells are expanded in the patient, which are able to recognize and killdiseased cells. When an immunoreactive cell of the invention, a T cellreceptor of the invention, an artificial T cell receptor of theinvention, a nucleic acid of the invention comprising a nucleotidesequence encoding a T cell receptor of the invention or encoding anartificial T cell receptor of the invention or a cell of the inventioncomprising a T cell receptor or an artificial T cell receptor of theinvention and/or comprising a nucleic acid of the invention comprising anucleotide sequence encoding a T cell receptor of the invention orencoding an artificial T cell receptor of the invention is administered,the treatment preferably involves a passive immunization. Preferably,CLDN6-specific T cells which are able to recognize and kill diseasedcells and which were optionally genetically engineered and/or expandedin vitro are adoptively transferred to a patient.

In one aspect, the invention relates to a pharmaceutical compositioncomprising one or more of:

(i) the peptide of the invention;

(ii) the nucleic acid of the invention;

(iii) the cell of the invention;

(iv) the immunoreactive cell of the invention;

(v) the binding agent of the invention;

(vi) the T cell receptor of the invention; and

(vi) the artificial T cell receptor of the invention.

A pharmaceutical composition of the invention may comprise apharmaceutically acceptable carrier and may optionally comprise one ormore adjuvants, stabilizers etc. The pharmaceutical composition may inthe form of a therapeutic or prophylactic vaccine. In one embodiment,the pharmaceutical composition is for use in treating or preventing acancer disease such as those described herein.

Administration of a pharmaceutical composition as described above mayprovide MHC class II-presented epitopes that are capable of eliciting aCD4+ helper T cell response and/or a CD8+ T cell response against CLDN6(including cells expressing CLDN6 on their surface and/or presentingCLDN6 in the context of MHC molecules). Alternatively or additionally,administration of a pharmaceutical composition as described above mayprovide MHC class I-presented epitopes that are capable of eliciting aCD8+ T cell response against CLDN6.

In a further aspect, the invention relates to a method of treating orpreventing a cancer disease comprising administering to a patient thepharmaceutical composition of the invention.

In a further aspect, the invention relates to the peptide of theinvention, the nucleic acid of the invention, the cell of the invention,the immunoreactive cell of the invention, the binding agent of theinvention, the T cell receptor of the invention, or the artificial Tcell receptor of the invention for use in therapy, in particular for usein treating or preventing cancer.

Another aspect relates to a method for inducing an immune response in asubject, comprising administering to the subject a pharmaceuticalcomposition of the invention.

Another aspect relates to a method for stimulating, priming and/orexpanding T cells, comprising contacting T cells with one or more of:the peptide of the invention, the nucleic acid of the inventioncomprising a nucleotide sequence encoding the peptide of the invention,the cell of the invention comprising said nucleic acid and/or the cellof the invention that presents the peptide of the invention or aprocession product thereof. In one embodiment, the peptide of theinvention is presented in the context of MHC molecules such as MHCmolecules on the surface of cells, e.g. antigen-presenting cells.

In this aspect, the invention may relate to a method for preparingCLDN6-specific T cells. The T cells may be stimulated, primed and/orexpanded in vitro or in vivo. Preferably, the T cells are present in asample obtained from a subject. The stimulated, primed and/or expanded Tcells may be administered to a subject and may be autologous,allogeneic, syngeneic to the subject.

The invention in the above aspects of a method for inducing an immuneresponse in a subject or of a method for stimulating, priming and/orexpanding T cells may relate to a method for treating cancer diseases ina subject.

Another aspect relates to a method of killing cancer cells in a subject,comprising the step of providing to the subject a therapeuticallyeffective amount of the peptide of the invention, the nucleic acid ofthe invention, the cell of the invention, the immunoreactive cell of theinvention, the binding agent of the invention, the T cell receptor ofthe invention, or the artificial T cell receptor of the invention.

The compositions and agents described herein are preferably capable ofinducing or promoting a cellular response, preferably cytotoxic T cellactivity, against a disease characterized by expression of CLDN6 and/orpresentation of CLDN6 with class I MHC, e.g. a cancer disease.

In one aspect, the invention provides the agents and compositionsdescribed herein for use in the methods of treatment described herein.

The treatments of cancer diseases described herein can be combined withsurgical resection and/or radiation and/or traditional chemotherapy.

In another aspect, the invention relates to a method for determining animmune response in a subject, comprising determining T cells reactivewith a peptide of the invention or a cell of the invention presenting apeptide of the invention or a procession product thereof in a biologicalsample isolated from the subject. The method may comprise the steps of:

(a) incubating a sample comprising T cells isolated from a subject withone or more of:

(i) the peptide of the invention;

(ii) the nucleic acid of the invention comprising a nucleotide sequenceencoding the peptide of the invention; and

(iii) the cell of the invention comprising said nucleic acid or the cellof the invention presenting a peptide of the invention or a processionproduct thereof;

and

(b) detecting the specific activation of the T cells, therefromdetermining the presence or absence of an immune response in saidsubject.

The invention in the above aspects of a method for determining an immuneresponse in a subject may relate to a method for diagnosing cancerdiseases in a subject.

In one embodiment of the methods for diagnosis, the biological sample isfrom a tissue or organ wherein the cells when the tissue or organ isdisease free do not substantially express CLDN6.

Typically, the level of T cells in a biological sample is compared to areference level, wherein a deviation from said reference level isindicative of the presence and/or stage of a disease in a subject. Thereference level may be a level as determined in a control sample (e.g.,from a healthy tissue or subject) or a median level from healthysubjects. A “deviation” from said reference level designates anysignificant change, such as an increase by at least 10%, 20%, or 30%,preferably by at least 40% or 50%, or even more. Preferably, thepresence of the T cells in said biological sample or a quantity of the Tcells in the biological sample which is increased compared to areference level indicates the presence of a disease.

T cells may be isolated from patient peripheral blood, lymph nodes,tissue samples such as derived from biopsy and resection, or othersource. Reactivity assays may be performed on primary T cells or otherappropriate derivatives. For example, T cells may be fused to generatehybridomas. Assays for measuring T cell responsiveness are known in theart, and include proliferation assays and cytokine release assays.

Assays and indices for detecting reactive T cells include but are notlimited to the use of IFNγ ELISPOT and IFNγ intracellular cytokinestaining. Other various methods are known in the art for determiningwhether a T cell clone will respond to a particular peptide. Typicallythe peptide is added to a suspension of the T cells for a period of fromone to three days. The response of the T cells may be measured byproliferation, e.g., uptake of labeled thymidine, or by release ofcytokines, e.g., IL-2. Various assays are available for detecting thepresence of released cytokines. T cell cytotoxic assays can be used todetect cytotoxic T cells having specificity for antigens. In oneembodiment, cytotoxic T cells are tested for their ability to killtarget cells presenting an antigen with MHC class I molecules. Targetcells presenting an antigen may be labeled and added to a suspension ofT cells from a patient sample. The cytotoxicity may be measured byquantifying the release of label from lysed cells. Controls forspontaneous and total release may be included in the assay.

In one embodiment of the invention, a cancer described herein involvescancer cells expressing CLDN6 and/or presenting CLDN6 in the context ofMHC molecules. In one embodiment of the invention, diseased cells arecancer cells. In one embodiment, diseased cells such as cancer cells arecells expressing CLDN6 and/or presenting CLDN6 in the context of MHCmolecules. In one embodiment, expression of CLDN6 is on the surface of adiseased cell.

In one embodiment of the invention, a cancer is selected from the groupconsisting of urinary bladder cancer, ovarian cancer, in particularovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer,including small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC), in particular squamous cell lung carcinoma and adenocarcinoma,gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skincancer, in particular basal cell carcinoma and squamous cell carcinoma,malignant melanoma, head and neck cancer, in particular malignantpleomorphic adenoma, sarcoma, in particular synovial sarcoma andcarcinosarcoma, bile duct cancer, cancer of the urinary bladder, inparticular transitional cell carcinoma and papillary carcinoma, kidneycancer, in particular renal cell carcinoma including clear cell renalcell carcinoma and papillary renal cell carcinoma, colon cancer, smallbowel cancer, including cancer of the ileum, in particular small boweladenocarcinoma and adenocarcinoma of the ileum, testicular embryonalcarcinoma, placental choriocarcinoma, cervical cancer, testicularcancer, in particular testicular seminoma, testicular teratoma andembryonic testicular cancer, uterine cancer, germ cell tumors such as ateratocarcinoma or an embryonal carcinoma, in particular germ celltumors of the testis, and the metastatic forms thereof.

In one embodiment of the invention, cancer cells are cancer cells of acancer selected from the group consisting of urinary bladder cancer,ovarian cancer, in particular ovarian adenocarcinoma and ovarianteratocarcinoma, lung cancer, including small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC), in particular squamous cell lungcarcinoma and adenocarcinoma, gastric cancer, breast cancer, hepaticcancer, pancreatic cancer, skin cancer, in particular basal cellcarcinoma and squamous cell carcinoma, malignant melanoma, head and neckcancer, in particular malignant pleomorphic adenoma, sarcoma, inparticular synovial sarcoma and carcinosarcoma, bile duct cancer, cancerof the urinary bladder, in particular transitional cell carcinoma andpapillary carcinoma, kidney cancer, in particular renal cell carcinomaincluding clear cell renal cell carcinoma and papillary renal cellcarcinoma, colon cancer, small bowel cancer, including cancer of theileum, in particular small bowel adenocarcinoma and adenocarcinoma ofthe ileum, testicular embryonal carcinoma, placental choriocarcinoma,cervical cancer, testicular cancer, in particular testicular seminoma,testicular teratoma and embryonic testicular cancer, uterine cancer,germ cell tumors such as a teratocarcinoma or an embryonal carcinoma, inparticular germ cell tumors of the testis, and the metastatic formsthereof.

According to the invention, CLDN6 preferably has the amino acid sequenceaccording to SEQ ID NO: 1 or 2.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,(1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of biochemistry, cell biology,immunology, and recombinant DNA techniques which are explained in theliterature in the field (cf., e.g., Molecular Cloning: A LaboratoryManual, 2^(nd) Edition, J. Sambrook et al. eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps although in some embodiments suchother member, integer or step or group of members, integers or steps maybe excluded, i.e. the subject-matter consists in the inclusion of astated member, integer or step or group of members, integers or steps.The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”), provided herein is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed. No language in the specification should be construedas indicating any non-claimed element essential to the practice of theinvention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

The term “recombinant” in the context of the present invention means“made through genetic engineering”. Preferably, a “recombinant object”such as a recombinant cell in the context of the present invention isnot occurring naturally.

The term “naturally occurring” as used herein refers to the fact that anobject can be found in nature. For example, a peptide or nucleic acidthat is present in an organism (including viruses) and can be isolatedfrom a source in nature and which has not been intentionally modified byman in the laboratory is naturally occurring.

The term “immune response” refers to an integrated bodily response to anantigen and preferably refers to a cellular immune response or acellular as well as a humoral immune response. The immune response maybe protective/preventive/prophylactic and/or therapeutic.

“Inducing an immune response” may mean that there was no immune responseagainst a particular antigen before induction, but it may also mean thatthere was a certain level of immune response against a particularantigen before induction and after induction said immune response isenhanced. Thus, “inducing an immune response” also includes “enhancingan immune response”. Preferably, after inducing an immune response in asubject, said subject is protected from developing a disease such as acancer disease or the disease condition is ameliorated by inducing animmune response. For example, an immune response against atumor-associated antigen such as CLDN6 may be induced in a patienthaving a cancer disease or in a subject being at risk of developing acancer disease. Inducing an immune response in this case may mean thatthe disease condition of the subject is ameliorated, that the subjectdoes not develop metastases, or that the subject being at risk ofdeveloping a cancer disease does not develop a cancer disease.

A “cellular immune response”, a “cellular response”, a “cellularresponse against an antigen” or a similar term is meant to include acellular response directed to cells characterized by presentation of anantigen with class I or class II MHC. The cellular response relates tocells called T cells or T-lymphocytes which act as either ‘helpers’ or‘killers’. The helper T cells (also termed CD4⁺ T cells) play a centralrole by regulating the immune response and the killer cells (also termedcytotoxic T cells, cytolytic T cells, CD8⁺ T cells or CTLs) killdiseased cells such as cancer cells, preventing the production of morediseased cells.

The term “antigen” relates to an agent comprising an epitope againstwhich an immune response is to be generated and/or is directed.Preferably, an antigen in the context of the present invention is amolecule which, optionally after processing, induces an immune reaction,which is preferably specific for the antigen or cells expressing and/orpresenting the antigen. The term “antigen” includes in particularproteins and peptides. An antigen is preferably a product whichcorresponds to or is derived from a naturally occurring antigen. Suchnaturally occurring antigens may include or may be derived fromtumor-associated antigens.

In particular, the antigen or peptide fragments thereof should berecognizable by a T cell receptor. Preferably, the antigen or peptide ifrecognized by a T cell receptor is able to induce in the presence ofappropriate co-stimulatory signals, clonal expansion of the T cellcarrying the T cell receptor recognizing the antigen or peptide. In thecontext of the embodiments of the present invention, the antigen ispreferably presented by a cell, preferably by an antigen presenting celland/or a diseased cell, in the context of MHC molecules, which mayresult in an immune reaction against the antigen (or cell presenting theantigen).

In a preferred embodiment, an antigen is a tumor-associated antigen,i.e., a constituent of cancer cells which may be derived from thecytoplasm, the cell surface and the cell nucleus, in particular thoseantigens which are produced, preferably in large quantity, intracellularor as surface antigens on cancer cells.

In the context of the present invention, the term “tumor-associatedantigen” or “tumor antigen” relates to proteins that are under normalconditions specifically expressed in a limited number of tissues and/ororgans or in specific developmental stages, for example, thetumor-associated antigen may be under normal conditions specificallyexpressed in stomach tissue, preferably in the gastric mucosa, inreproductive organs, e.g., in testis, in trophoblastic tissue, e.g., inplacenta, or in germ line cells, and are expressed or aberrantlyexpressed in one or more tumor or cancer tissues. In this context, “alimited number” preferably means not more than 3, more preferably notmore than 2. The tumor-associated antigens in the context of the presentinvention include, for example, differentiation antigens, preferablycell type specific differentiation antigens, i.e., proteins that areunder normal conditions specifically expressed in a certain cell type ata certain differentiation stage, cancer/testis antigens, i.e., proteinsthat are under normal conditions specifically expressed in testis andsometimes in placenta, and germ line specific antigens. In the contextof the present invention, the tumor-associated antigen is preferablyassociated with the cell surface of a cancer cell and is preferably notor only rarely expressed in normal tissues. Preferably, thetumor-associated antigen or the aberrant expression of thetumor-associated antigen identifies cancer cells. In the context of thepresent invention, the tumor-associated antigen that is expressed by acancer cell in a subject, e.g., a patient suffering from a cancerdisease, is preferably a self-protein in said subject. In preferredembodiments, the tumor-associated antigen in the context of the presentinvention is expressed under normal conditions specifically in a tissueor organ that is non-essential, i.e., tissues or organs which whendamaged by the immune system do not lead to death of the subject, or inorgans or structures of the body which are not or only hardly accessibleby the immune system. Preferably, the amino acid sequence of thetumor-associated antigen is identical between the tumor-associatedantigen which is expressed in normal tissues and the tumor-associatedantigen which is expressed in cancer tissues. Preferably, atumor-associated antigen is presented by a cancer cell in which it isexpressed.

Various aspects of the invention involve the tumor-associated antigenCLDN6 and the present invention may involve the stimulation or provisionof an anti-tumor CTL reaction against cancer cells expressing saidtumor-associated antigen and preferably presenting said tumor-associatedantigen with class I MHC.

Claudins are a family of proteins that are the most important componentsof tight junctions, where they establish the paracellular barrier thatcontrols the flow of molecules in the intercellular space between cellsof an epithelium. Claudins are transmembrane proteins spanning themembrane 4 times with the N-terminal and the C-terminal end both locatedin the cytoplasm. The first extracellular loop, termed EC1 or ECL1,consists on average of 53 amino acids, and the second extracellularloop, termed EC2 or ECL2, consists of around 24 amino acids. Cellsurface proteins of the claudin family, such as CLDN6, are expressed intumors of various origins, and are particularly suited as targetstructures in connection with antibody-mediated cancer immunotherapy dueto their selective expression (no expression in a toxicity relevantnormal tissue) and localization to the plasma membrane.

CLDN6 has been identified as differentially expressed in tumor tissues,with the only normal tissues expressing CLDN6 being placenta.

CLDN6 has been found to be expressed, for example, in ovarian cancer,lung cancer, gastric cancer, breast cancer, hepatic cancer, pancreaticcancer, skin cancer, melanomas, head neck cancer, sarcomas, bile ductcancer, renal cell cancer, and urinary bladder cancer. CLDN6 is aparticularly preferred target for the prevention and/or treatment ofovarian cancer, in particular ovarian adenocarcinoma and ovarianteratocarcinoma, lung cancer, including small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC), in particular squamous cell lungcarcinoma and adenocarcinoma, gastric cancer, breast cancer, hepaticcancer, pancreatic cancer, skin cancer, in particular basal cellcarcinoma and squamous cell carcinoma, malignant melanoma, head and neckcancer, in particular malignant pleomorphic adenoma, sarcoma, inparticular synovial sarcoma and carcinosarcoma, bile duct cancer, cancerof the urinary bladder, in particular transitional cell carcinoma andpapillary carcinoma, kidney cancer, in particular renal cell carcinomaincluding clear cell renal cell carcinoma and papillary renal cellcarcinoma, colon cancer, small bowel cancer, including cancer of theileum, in particular small bowel adenocarcinoma and adenocarcinoma ofthe ileum, testicular embryonal carcinoma, placental choriocarcinoma,cervical cancer, testicular cancer, in particular testicular seminoma,testicular teratoma and embryonic testicular cancer, uterine cancer,germ cell tumors such as a teratocarcinoma or an embryonal carcinoma, inparticular germ cell tumors of the testis, and the metastatic formsthereof. In one embodiment, the cancer disease associated with CLDN6expression is selected from the group consisting of ovarian cancer, lungcancer, metastatic ovarian cancer and metastatic lung cancer.Preferably, the ovarian cancer is a carcinoma or an adenocarcinoma.Preferably, the lung cancer is a carcinoma or an adenocarcinoma, andpreferably is bronchiolar cancer such as a bronchiolar carcinoma orbronchiolar adenocarcinoma.

The term “CLDN” as used herein means claudin and includes CLDN6.Preferably, a claudin is a human claudin. The term “CLDN6” relates toclaudin 6 and includes any variants thereof.

The term “CLDN6” preferably relates to human CLDN6, and, in particular,to a protein comprising, preferably consisting of the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the sequence listing or avariant of said amino acid sequence. The first extracellular loop ofCLDN6 preferably comprises amino acids 28 to 80, more preferably aminoacids 28 to 76 of the amino acid sequence shown in SEQ ID NO: 1 or theamino acid sequence shown in SEQ ID NO: 2. The second extracellular loopof CLDN6 preferably comprises amino acids 138 to 160, preferably aminoacids 141 to 159, more preferably amino acids 145 to 157 of the aminoacid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown inSEQ ID NO: 2. Said first and second extracellular loops preferably formthe extracellular portion of CLDN6.

The term “variant” according to the invention refers, in particular, tomutants, splice variants, conformations, isoforms, allelic variants,species variants and species homologs, in particular those which arenaturally present. An allelic variant relates to an alteration in thenormal sequence of a gene, the significance of which is often unclear.Complete gene sequencing often identifies numerous allelic variants fora given gene. A species homolog is a nucleic acid or amino acid sequencewith a different species of origin from that of a given nucleic acid oramino acid sequence. The term “variant” shall encompass anyposttranslationally modified variants and conformation variants.

According to the various aspects of the invention, the aim is preferablyto induce or determine an immune response against cancer cellsexpressing CLDN6 and preferably being characterized by presentation ofCLDN6, and to diagnose, treat or prevent a cancer disease involvingcells expressing CLDN6. Preferably the immune response involves thestimulation of an anti-CLDN6 CTL response against cancer cellsexpressing CLDN6 and preferably presenting CLDN6 with class I MHC.

According to the invention, the term “CLDN6 positive cancer” or similarterms means a cancer involving cancer cells expressing CLDN6, preferablyon the surface of said cancer cells.

Alternatively or additionally, said cancer cells expressing CLDN6present CLDN6 in the context of MHC molecules. Cancer cells presentingCLDN6 in the context of MHC molecules can be targeted by immunoreactivecells carrying T cell receptors while cancer cells expressing CLDN6 onthe surface can be targeted by immunoreactive cells carrying artificialT cell receptors.

“Cell surface” is used in accordance with its normal meaning in the art,and thus includes the outside of the cell which is accessible to bindingby proteins and other molecules

CLDN6 is expressed on the surface of cells if it is located at thesurface of said cells and is accessible to binding by CLDN6-specificantibodies added to the cells.

The term “extracellular portion” or “exodomain” in the context of thepresent invention refers to a part of a molecule such as a protein thatis facing the extracellular space of a cell and preferably is accessiblefrom the outside of said cell, e.g., by antigen-binding molecules suchas antibodies located outside the cell. Preferably, the term refers toone or more extracellular loops or domains or a fragment thereof.

The term “portion” refers to a fraction. With respect to a particularstructure such as an amino acid sequence or protein the term “portion”thereof may designate a continuous or a discontinuous fraction of saidstructure. Preferably, a portion of an amino acid sequence comprises atleast 1%, at least 5%, at least 10%, at least 20%, at least 30%,preferably at least 40%, preferably at least 50%, more preferably atleast 60%, more preferably at least 70%, even more preferably at least80%, and most preferably at least 90% of the amino acids of said aminoacid sequence. Preferably, if the portion is a discontinuous fractionsaid discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or moreparts of a structure, each part being a continuous element of thestructure. For example, a discontinuous fraction of an amino acidsequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, preferably notmore than 4 parts of said amino acid sequence, wherein each partpreferably comprises at least 5 continuous amino acids, at least 10continuous amino acids, preferably at least 20 continuous amino acids,preferably at least 30 continuous amino acids of the amino acidsequence.

The terms “part” and “fragment” are used interchangeably herein andrefer to a continuous element. For example, a part of a structure suchas an amino acid sequence or protein refers to a continuous element ofsaid structure. A portion, a part or a fragment of a structurepreferably comprises one or more functional properties of saidstructure. For example, a portion, a part or a fragment of an epitope,peptide or protein is preferably immunologically equivalent to theepitope, peptide or protein it is derived from. In the context of thepresent invention, a “part” of a structure such as an amino acidsequence preferably comprises, preferably consists of at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 98%, at least 99% of the entirestructure or amino acid sequence. A part or fragment of a proteinsequence preferably comprises a sequence of at least 6, in particular atleast 8, at least 12, at least 15, at least 20, at least 30, at least50, or at least 100 consecutive amino acids of the protein sequence.Portions, parts or fragments as discussed above are encompassed by theterm “variant” used herein.

According to the invention, CLDN6 is not substantially expressed in acell if the level of expression is lower compared to expression inplacenta cells or placenta tissue. Preferably, the level of expressionis less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or0.05% of the expression in placenta cells or placenta tissue or evenlower. Preferably, CLDN6 is not substantially expressed in a cell if thelevel of expression exceeds the level of expression in non-canceroustissue other than placenta by no more than 2-fold, preferably 1.5-fold,and preferably does not exceed the level of expression in saidnon-cancerous tissue. Preferably, CLDN6 is not substantially expressedin a cell if the level of expression is below the detection limit and/orif the level of expression is too low to allow binding by CLDN6-specificantibodies added to the cells.

According to the invention, CLDN6 is expressed in a cell if the level ofexpression exceeds the level of expression in non-cancerous tissue otherthan placenta preferably by more than 2-fold, preferably 10-fold,100-fold, 1000-fold, or 10000-fold. Preferably, CLDN6 is expressed in acell if the level of expression is above the detection limit and/or ifthe level of expression is high enough to allow binding byCLDN6-specific antibodies added to the cells. Preferably, CLDN6expressed in a cell is expressed or exposed on the surface of said cell.

“Target cell” shall mean a cell which is a target for an immune responsesuch as a cellular immune response. Target cells include cells thatpresent an antigen or an antigen epitope, i.e. a peptide fragmentderived from an antigen, and include any undesirable cell such as acancer cell.

In preferred embodiments, the target cell is a cell expressing CLDN6which preferably is present on the cell surface and/or presented withclass I MHC.

The term “epitope” refers to an antigenic determinant in a molecule suchas an antigen, i.e., to a part in or fragment of the molecule that isrecognized by the immune system, for example, that is recognized by a Tcell, in particular when presented in the context of MHC molecules. Anepitope of a protein such as a tumor-associated antigen preferablycomprises a continuous or discontinuous portion of said protein and ispreferably between 5 and 100, preferably between 5 and 50, morepreferably between 8 and 30, most preferably between 10 and 25 aminoacids in length, for example, the epitope may be preferably 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids inlength. It is particularly preferred that the epitope in the context ofthe present invention is a T cell epitope.

Terms such as “epitope”, “antigen fragment”, “antigen peptide” or“immunogenic peptide” are used interchangeably herein and preferablyrelate to an incomplete representation of an antigen which is preferablycapable of eliciting an immune response against the antigen or a cellexpressing or comprising and preferably presenting the antigen.Preferably, the terms relate to an immunogenic portion of an antigen.Preferably, it is a portion of an antigen that is recognized (i.e.,specifically bound) by a T cell receptor, in particular if presented inthe context of MHC molecules. Certain preferred immunogenic portionsbind to an MHC class I or class II molecule such as on the surface of acell and thus are MHC binding peptides. As used herein, a peptide issaid to “bind to” an MHC class I or class II molecule if such binding isdetectable using any assay known in the art.

Preferably, the peptides disclosed herein comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 3, 4 and 5 ora variant of said amino acid sequence are capable of stimulating animmune response, preferably a cellular response against CLDN6 or cellscharacterized by expression of CLDN6 and preferably characterized bypresentation of CLDN6. Preferably, such peptide is capable ofstimulating a cellular response against a cell characterized bypresentation of CLDN6 with class I MHC and preferably is capable ofstimulating CLDN6-responsive CTL. Preferably, the peptides according tothe invention are MHC class I and/or class II presented peptides or canbe processed to produce MHC class I and/or class II presented peptides.Preferably, the sequence bound to the MHC molecule is selected from SEQID NOs: 3, 4 and 5.

If an antigen peptide is to be presented directly, i.e. withoutprocessing, in particular without cleavage, it has a length which issuitable for binding to an MHC molecule, in particular a class I MHCmolecule, and preferably is 7-20 amino acids in length, more preferably7-12 amino acids in length, more preferably 8-11 amino acids in length,in particular 9 or 10 amino acids in length. Preferably the sequence ofan antigen peptide which is to be presented directly substantiallycorresponds and is preferably completely identical to a sequenceselected from SEQ ID NOs: 3, 4 and 5.

If an antigen peptide is to be presented following processing, inparticular following cleavage, the peptide produced by processing has alength which is suitable for binding to an MHC molecule, in particular aclass I MHC molecule, and preferably is 7-20 amino acids in length, morepreferably 7-12 amino acids in length, more preferably 8-11 amino acidsin length, in particular 9 or 10 amino acids in length. Preferably, thesequence of the peptide which is to be presented following processingsubstantially corresponds and is preferably completely identical to asequence selected from SEQ ID NOs: 3, 4 and 5. Thus, an antigen peptideaccording to the invention in one embodiment comprises a sequenceselected from SEQ ID NOs: 3, 4 and 5 and following processing of theantigen peptide makes up a sequence selected from SEQ ID NOs: 3, 4 and5.

Peptides having amino acid sequences substantially corresponding to asequence of a peptide which is presented by MHC molecules may differ atone or more residues that are not essential for TCR recognition of thepeptide as presented by the MHC, or for peptide binding to MHC. Suchsubstantially corresponding peptides preferably are also capable ofstimulating an antigen-specific cellular response such asantigen-specific CTL. Peptides having amino acid sequences differingfrom a presented peptide at residues that do not affect TCR recognitionbut improve the stability of binding to MHC may improve theimmunogenicity of the antigen peptide, and may be referred to herein as“optimized peptides”. Using existing knowledge about which of theseresidues may be more likely to affect binding either to the MHC or tothe TCR, a rational approach to the design of substantiallycorresponding peptides may be employed. Resulting peptides that arefunctional are contemplated as antigen peptides. Sequences as discussedabove are encompassed by the term “variant” used herein.

“Antigen processing” refers to the degradation of an antigen intoprocession products, which are fragments of said antigen (e.g., thedegradation of a protein into peptides) and the association of one ormore of these fragments (e.g., via binding) with MHC molecules forpresentation by cells, preferably antigen presenting cells to specific Tcells.

An antigen-presenting cell (APC) is a cell that displays antigen in thecontext of major histocompatibility complex (MHC) on its surface. Tcells may recognize this complex using their T cell receptor (TCR).Antigen-presenting cells process antigens and present them to T cells.

Professional antigen-presenting cells are very efficient atinternalizing antigen, either by phagocytosis or by receptor-mediatedendocytosis, and then displaying a fragment of the antigen, bound to aclass II MHC molecule, on their membrane. The T cell recognizes andinteracts with the antigen-class II MHC molecule complex on the membraneof the antigen-presenting cell. An additional co-stimulatory signal isthen produced by the antigen-presenting cell, leading to activation ofthe T cell. The expression of co-stimulatory molecules is a definingfeature of professional antigen-presenting cells. Antigen-presentingcells include professional antigen-presenting cells and non-professionalantigen-presenting cells.

The main types of professional antigen-presenting cells are dendriticcells, which have the broadest range of antigen presentation, and areprobably the most important antigen-presenting cells, macrophages,B-cells, and certain activated epithelial cells.

Non-professional antigen-presenting cells do not constitutively expressthe MHC class II proteins required for interaction with naive T cells;these are expressed only upon stimulation of the non-professionalantigen-presenting cells by certain cytokines such as IFNγ.

Dendritic cells (DCs) are leukocyte populations that present antigenscaptured in peripheral tissues to T cells via both MHC class II and Iantigen presentation pathways. It is well known that dendritic cells arepotent inducers of immune responses and the activation of these cells isa critical step for the induction of antitumoral immunity.

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFa to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which can be used as a simple way to discriminate between twowell characterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation.

Immature dendritic cells are characterized as antigen presenting cellswith a high capacity for antigen uptake and processing, which correlateswith the high expression of Fcγ receptor and mannose receptor. Themature phenotype is typically characterized by a lower expression ofthese markers, but a high expression of cell surface moleculesresponsible for T cell activation such as class I and class II MHC,adhesion molecules (e.g. CD54 and CD11) and costimulatory molecules(e.g., CD40, CD80, CD86 and 4-1 BB).

Dendritic cell maturation is referred to as the status of dendritic cellactivation at which such antigen-presenting dendritic cells lead to Tcell priming, while presentation by immature dendritic cells results intolerance. Dendritic cell maturation is chiefly caused by biomoleculeswith microbial features detected by innate receptors (bacterial DNA,viral RNA, endotoxin, etc.), pro-inflammatory cytokines (TNF, IL-1,IFNs), ligation of CD40 on the dendritic cell surface by CD40L, andsubstances released from cells undergoing stressful cell death. Thedendritic cells can be derived by culturing bone marrow cells in vitrowith cytokines, such as granulocyte-macrophage colony-stimulating factor(GM-CSF) and tumor necrosis factor alpha.

Cells such as antigen presenting cells or target cells can be loadedwith MHC class I presented peptides by exposing, i.e. pulsing, the cellswith the peptide or transducing the cells with nucleic acid, preferablyRNA, encoding a peptide or protein comprising the peptide to bepresented, e.g. a nucleic acid encoding the antigen.

In some embodiments, a pharmaceutical composition of the inventioncomprises an antigen presenting cell loaded with antigen peptide. Inthis respect, protocols may rely on in vitro culture/differentiation ofdendritic cells manipulated in such a way that they artificially presentantigen peptide. Production of genetically engineered dendritic cellsmay involve introduction of nucleic acids encoding antigens or antigenpeptides into dendritic cells. Transfection of dendritic cells with mRNAis a promising antigen-loading technique of stimulating strong antitumorimmunity. Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes. Alternatively, a gene delivery vehicle thattargets a dendritic or other antigen presenting cell may be administeredto a patient, resulting in transfection that occurs in vivo. In vivo andex vivo transfection of dendritic cells, for example, may generally beperformed using any methods known in the art, such as those described inWO 97/24447, or the gene gun approach described by Mahvi et al.,Immunology and cell Biology 75: 456-460, 1997. Antigen loading ofdendritic cells may be achieved by incubating dendritic cells orprogenitor cells with antigen, DNA (naked or within a plasmid vector) orRNA; or with antigen-expressing recombinant bacteria or viruses (e.g.,vaccinia, fowipox, adenovirus or lentivirus vectors).

The term “immunogenicity” relates to the relative efficiency of anantigen to induce an immune reaction.

The term “immune effector functions” in the context of the presentinvention includes any functions mediated by components of the immunesystem that result, for example, in the killing of tumor cells, or inthe inhibition of tumor growth and/or inhibition of tumor development,including inhibition of tumor dissemination and metastasis. Preferably,the immune effector functions in the context of the present inventionare T cell mediated effector functions. Such functions comprise in thecase of a helper T cell (CD4⁺ T cell) the recognition of an antigen oran antigen peptide derived from an antigen in the context of MHC classII molecules by T cell receptors, the release of cytokines and/or theactivation of CD8⁺ lymphocytes (CTLs) and/or B-cells, and in the case ofCTL the recognition of an antigen or an antigen peptide derived from anantigen in the context of MHC class I molecules by T cell receptors, theelimination of cells presented in the context of MHC class I molecules,i.e., cells characterized by presentation of an antigen with class IMHC, for example, via apoptosis or perforin-mediated cell lysis,production of cytokines such as IFN-γ and TNF-α, and specific cytolytickilling of antigen expressing target cells.

The term “immunoreactive cell” or “immune effector cell” in the contextof the present invention relates to a cell which exerts effectorfunctions during an immune reaction. An “immunoreactive cell” preferablyis capable of binding an antigen such as an antigen expressed on thesurface of a cell or a cell characterized by presentation of an antigenor an antigen peptide derived from an antigen and mediating an immuneresponse. For example, such cells secrete cytokines and/or chemokines,kill microbes, secrete antibodies, recognize infected or cancerouscells, and optionally eliminate such cells. For example, immunoreactivecells comprise T cells (cytotoxic T cells, helper T cells, tumorinfiltrating T cells), B cells, natural killer cells, neutrophils,macrophages, and dendritic cells. Preferably, in the context of thepresent invention, “immunoreactive cells” are T cells, preferably CD4⁺and/or CD8⁺ T cells.

Preferably, an “immunoreactive cell” recognizes an antigen or an antigenpeptide derived from an antigen with some degree of specificity, inparticular if presented in the context of MHC molecules such as on thesurface of antigen presenting cells or diseased cells such as cancercells. Preferably, said recognition enables the cell that recognizes anantigen or an antigen peptide derived from said antigen to be responsiveor reactive. If the cell is a helper T cell (CD4⁺ T cell) bearingreceptors that recognize an antigen or an antigen peptide derived froman antigen in the context of MHC class II molecules such responsivenessor reactivity may involve the release of cytokines and/or the activationof CD8⁺ lymphocytes (CTLs) and/or B-cells. If the cell is a CTL suchresponsiveness or reactivity may involve the elimination of cellspresented in the context of MHC class I molecules, i.e., cellscharacterized by presentation of an antigen with class I MHC, forexample, via apoptosis or perforin-mediated cell lysis. According to theinvention, CTL responsiveness may include sustained calcium flux, celldivision, production of cytokines such as IFN-γ and TNF-α, up-regulationof activation markers such as CD44 and CD69, and specific cytolytickilling of antigen expressing target cells. CTL responsiveness may alsobe determined using an artificial reporter that accurately indicates CTLresponsiveness. Such CTL that recognizes an antigen or an antigenpeptide derived from an antigen and are responsive or reactive are alsotermed “antigen-responsive CTL” herein. If the cell is a B cell suchresponsiveness may involve the release of immunoglobulins.

According to the invention, the term “immunoreactive cell” also includesa cell which can mature into an immune cell (such as T cell, inparticular T helper cell, or cytolytic T cell) with suitable stimulationImmunoreactive cells comprise CD34⁺ hematopoietic stem cells, immatureand mature T cells and immature and mature B cells. If production ofcytolytic or T helper cells recognizing an antigen is desired, theimmunoreactive cell is contacted with a cell presenting an antigen orantigen peptide under conditions which favor production, differentiationand/or selection of cytolytic T cells and of T helper cells. Thedifferentiation of T cell precursors into a cytolytic T cell, whenexposed to an antigen, is similar to clonal selection of the immunesystem.

A “lymphoid cell” is a cell which, optionally after suitablemodification, e.g. after transfer of a T cell receptor, is capable ofproducing an immune response such as a cellular immune response, or aprecursor cell of such cell, and includes lymphocytes, preferably Tlymphocytes, lymphoblasts, and plasma cells. A lymphoid cell may be animmunoreactive cell as described herein. A preferred lymphoid cell is aT cell lacking endogenous expression of a T cell receptor and which canbe modified to express such T cell receptor on the cell surface.

The terms “T cell” and “T lymphocyte” are used interchangeably hereinand include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs,CD8+ T cells) which comprise cytolytic T cells.

T cells belong to a group of white blood cells known as lymphocytes, andplay a central role in cell-mediated immunity. They can be distinguishedfrom other lymphocyte types, such as B cells and natural killer cells bythe presence of a special receptor on their cell surface called T cellreceptors (TCR). The thymus is the principal organ responsible for the Tcell's maturation of T cells. Several different subsets of T cells havebeen discovered, each with a distinct function.

T helper cells assist other white blood cells in immunologic processes,including maturation of B cells into plasma cells and activation ofcytotoxic T cells and macrophages, among other functions. These cellsare also known as CD4+ T cells because they express the CD4 protein ontheir surface. Helper T cells become activated when they are presentedwith peptide antigens by MHC class II molecules that are expressed onthe surface of antigen presenting cells (APCs). Once activated, theydivide rapidly and secrete small proteins called cytokines that regulateor assist in the active immune response.

Cytotoxic T cells destroy virally infected cells and tumor cells, andare also implicated in transplant rejection. These cells are also knownas CD8+ T cells since they express the CD8 glycoprotein at theirsurface. These cells recognize their targets by binding to antigenassociated with MHC class I, which is present on the surface of nearlyevery cell of the body.

A majority of T cells have a T cell receptor (TCR) existing as a complexof several proteins. The actual T cell receptor is composed of twoseparate peptide chains, which are produced from the independent T cellreceptor alpha and beta (TCRα and TCRβ) genes and are called α- andβ-TCR chains. γδ T cells (gamma delta T cells) represent a small subsetof T cells that possess a distinct T cell receptor (TCR) on theirsurface. However, in γδ T cells, the TCR is made up of one γ-chain andone δ-chain. This group of T cells is much less common (2% of total Tcells) than the αβ T cells.

The structure of the T cell receptor is very similar to immunoglobulinFab fragments, which are regions defined as the combined light and heavychain of an antibody arm. Each chain of the TCR is a member of theimmunoglobulin superfamily and possesses one N-terminal immunoglobulin(Ig)-variable (V) domain, one Ig-constant (C) domain, atransmembrane/cell membrane-spanning region, and a short cytoplasmictail at the C-terminal end.

According to the invention, the term “variable region of a T cellreceptor” relates to the variable domains of the TCR chains.

The variable region of both the TCR α-chain and β-chain have threehypervariable or complementarity determining regions (CDRs), whereas thevariable region of the β-chain has an additional area ofhypervariability (HV4) that does not normally contact antigen andtherefore is not considered a CDR. CDR3 is the main CDR responsible forrecognizing processed antigen, although CDR1 of the α-chain has alsobeen shown to interact with the N-terminal part of the antigenicpeptide, whereas CDR1 of the β-chain interacts with the C-terminal partof the peptide. CDR2 is thought to recognize the MHC. CDR4 of theβ-chain is not thought to participate in antigen recognition, but hasbeen shown to interact with superantigens.

According to the invention, the term “at least one of the CDR sequences”preferably means at least the CDR3 sequence. The term “CDR sequences ofa T cell receptor chain” preferably relates to CDR1, CDR2 and CDR3 ofthe α-chain or β-chain of a T cell receptor.

The constant domain of the TCR domain consists of short connectingsequences in which a cysteine residue forms disulfide bonds, which formsa link between the two chains.

All T cells originate from hematopoietic stem cells in the bone marrow.Hematopoietic progenitors derived from hematopoietic stem cells populatethe thymus and expand by cell division to generate a large population ofimmature thymocytes. The earliest thymocytes express neither CD4 norCD8, and are therefore classed as double-negative (CD4−CD8−) cells. Asthey progress through their development they become double-positivethymocytes (CD4+CD8+), and finally mature to single-positive (CD4+CD8−or CD4−CD8+) thymocytes that are then released from the thymus toperipheral tissues.

The first signal in activation of T cells is provided by binding of theT cell receptor to a short peptide presented by the majorhistocompatibility complex (MHC) on another cell. This ensures that onlya T cell with a TCR specific to that peptide is activated. The partnercell is usually a professional antigen presenting cell (APC), usually adendritic cell in the case of naïve responses, although B cells andmacrophages can be important APCs. The peptides presented to CD8+ Tcells by MHC class I molecules are 8-10 amino acids in length; thepeptides presented to CD4+ T cells by MHC class II molecules are longer,as the ends of the binding cleft of the MHC class II molecule are open.

T cells may generally be prepared in vitro or ex vivo, using standardprocedures. For example, T cells may be present within (or isolatedfrom) bone marrow, peripheral blood or a fraction of bone marrow orperipheral blood of a mammal, such as a patient, using a commerciallyavailable cell separation system. Alternatively, T cells may be derivedfrom related or unrelated humans, non-human animals, cell lines orcultures. A “sample comprising T cells” may, for example, be peripheralblood mononuclear cells (PBMC).

T cells may be stimulated with antigen, peptide, nucleic acid and/orantigen presenting cells (APCs) that express an antigen. Suchstimulation is performed under conditions and for a time sufficient topermit the generation of T cells that are specific for an antigen, apeptide and/or cells presenting an antigen or a peptide.

Specific activation of CD4+ or CD8+ T cells may be detected in a varietyof ways. Methods for detecting specific T cell activation includedetecting the proliferation of T cells, the production of cytokines(e.g., lymphokines), or the generation of cytolytic activity. For CD4+ Tcells, a preferred method for detecting specific T cell activation isthe detection of the proliferation of T cells. For CD8+ T cells, apreferred method for detecting specific T cell activation is thedetection of the generation of cytolytic activity.

In order to generate CD8+ T cell lines, antigen-presenting cells,preferably autologous antigen-presenting cells, transfected with anucleic acid which produces the antigen may be used as stimulator cells.

Nucleic acids such as RNA encoding T cell receptor (TCR) chains may beintroduced into lymphoid cells such as T cells or other cells with lyticpotential. In a suitable embodiment, the TCR α- and β-chains are clonedout from an antigen-specific T cell line and used for adoptive T celltherapy. In this respect, the present invention provides T cellreceptors specific for CLDN6 or CLDN6 peptides disclosed herein. Ingeneral, this aspect of the invention relates to T cell receptors whichrecognize or bind CLDN6 peptides presented in the context of MHC. Thenucleic acids encoding α- and β-chains of a T cell receptor, e.g. a Tcell receptor provided according to the present invention, may becontained on separate nucleic acid molecules such as expression vectorsor alternatively, on a single nucleic acid molecule. Accordingly, theterm “a nucleic acid encoding a T cell receptor” or similar terms relateto nucleic acid molecules encoding the T cell receptor chains on thesame or preferably on different nucleic acid molecules.

The term “immunoreactive cell reactive with a peptide” relates to animmunoreactive cell which when it recognizes the peptide, in particularif presented in the context of MHC molecules such as on the surface ofantigen presenting cells or diseased cells such as cancer cells, exertseffector functions of immunoreactive cells as described above.

The term “T cell receptor reactive with a peptide” relates to a T cellreceptor which when present on an immunoreactive cell recognizes thepeptide, in particular if presented in the context of MHC molecules suchas on the surface of antigen presenting cells or diseased cells such ascancer cells, such that the immunoreactive cell exerts effectorfunctions of immunoreactive cells as described above.

The term “antigen-reactive T cell” or similar terms relate to a T cellwhich recognizes an antigen if presented in the context of MHC moleculessuch as on the surface of antigen presenting cells or diseased cellssuch as cancer cells and exerts effector functions of T cells asdescribed above.

The term “antigen-specific lymphoid cell” relates to a lymphoid cellwhich, in particular when provided with an antigen-specific T cellreceptor, recognizes the antigen if presented in the context of MHCmolecules such as on the surface of antigen presenting cells or diseasedcells such as cancer cells and preferably exerts effector functions of Tcells as described above. T cells and other lymphoid cells areconsidered to be specific for antigen if the cells kill target cellsexpressing an antigen and/or presenting an antigen peptide. T cellspecificity may be evaluated using any of a variety of standardtechniques, for example, within a chromium release assay orproliferation assay. Alternatively, synthesis of lymphokines (such asinterferon-γ) can be measured

The term “major histocompatibility complex” and the abbreviation “MHC”include MHC class I and MHC class II molecules and relate to a complexof genes which occurs in all vertebrates. MHC proteins or molecules areimportant for signaling between lymphocytes and antigen presenting cellsor diseased cells in immune reactions, wherein the MHC proteins ormolecules bind peptides and present them for recognition by T cellreceptors. The proteins encoded by the MHC are expressed on the surfaceof cells, and display both self antigens (peptide fragments from thecell itself) and nonself antigens (e.g., fragments of invadingmicroorganisms) to a T cell.

The MHC region is divided into three subgroups, class I, class II, andclass III. MHC class I proteins contain an α-chain and β2-microglobulin(not part of the MHC encoded by chromosome 15). They present antigenfragments to cytotoxic T cells. On most immune system cells,specifically on antigen-presenting cells, MHC class II proteins containα- and β-chains and they present antigen fragments to T-helper cells.MHC class III region encodes for other immune components, such ascomplement components and some that encode cytokines.

In humans, genes in the MHC region that encode antigen-presentingproteins on the cell surface are referred to as human leukocyte antigen(HLA) genes. However the abbreviation MHC is often used to refer to HLAgene products. HLA genes include the nine so-called classical MHC genes:HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA,and HLA-DRB1.

In one preferred embodiment of all aspects of the invention an MHCmolecule is an HLA molecule.

By “cell characterized by presentation of an antigen”, “cell presentingan antigen”, “antigen presented by a cell”, “antigen presented” orsimilar expressions is meant a cell such as a diseased cell such as acancer cell, or an antigen presenting cell presenting the antigen itexpresses or a fragment derived from said antigen, e.g. by processing ofthe antigen, in the context of MHC molecules, in particular MHC Class Imolecules. Similarly, the terms “disease characterized by presentationof an antigen” denotes a disease involving cells characterized bypresentation of an antigen, in particular with class I MHC. Presentationof an antigen by a cell may be effected by transfecting the cell with anucleic acid such as RNA encoding the antigen.

By “fragment of an antigen which is presented” or similar expressions ismeant that the fragment can be presented by MHC class I or class II,preferably MHC class I, e.g. when added directly to antigen presentingcells. In one embodiment, the fragment is a fragment which is naturallypresented by cells expressing an antigen.

Some therapeutic methods are based on a reaction of the immune system ofa patient, which results in a lysis of diseased cells which present anantigen with class I MHC. In this connection, for example autologouscytotoxic T lymphocytes specific for a complex of an antigen peptide andan MHC molecule may be administered to a patient having a disease. Theproduction of such cytotoxic T lymphocytes in vitro is known. An exampleof a method of differentiating T cells can be found in WO-A-9633265.Generally, a sample containing cells such as blood cells is taken fromthe patient and the cells are contacted with a cell which presents thecomplex and which can cause propagation of cytotoxic T lymphocytes (e.g.dendritic cells). The target cell may be a transfected cell such as aCOS cell. These transfected cells present the desired complex on theirsurface and, when contacted with cytotoxic T lymphocytes, stimulatepropagation of the latter. The clonally expanded autologous cytotoxic Tlymphocytes are then administered to the patient.

In another method of selecting cytotoxic T lymphocytes, fluorogenictetramers of MHC class I molecule/peptide complexes are used forobtaining specific clones of cytotoxic T lymphocytes (Altman et al.(1996), Science 274:94-96; Dunbar et al. (1998), Curr. Biol. 8:413-416,1998).

Furthermore, cells presenting the desired complex (e.g. dendritic cells)may be combined with cytotoxic T lymphocytes of healthy individuals oranother species (e.g. mouse) which may result in propagation of specificcytotoxic T lymphocytes with high affinity. The high affinity T cellreceptor of these propagated specific T lymphocytes may be cloned andoptionally humanized to a different extent, and the T cell receptorsthus obtained then transduced via gene transfer, for example usingretroviral vectors, into T cells of patients. Adoptive transfer may thenbe carried out using these genetically altered T lymphocytes(Stanislawski et al. (2001), Nat Immunol. 2:962-70; Kessels et al.(2001), Nat Immunol. 2:957-61.

Cytotoxic T lymphocytes may also be generated in vivo in a manner knownper se. One method uses nonproliferative cells expressing an MHC classI/peptide complex. The cells used here will be those which usuallyexpress the complex, such as irradiated tumor cells or cells transfectedwith one or both genes necessary for presentation of the complex (i.e.the antigenic peptide and the presenting MHC molecule). Anotherpreferred form is the introduction of an antigen in the form ofrecombinant RNA which may be introduced into cells by liposomal transferor by electroporation, for example. The resulting cells present thecomplex of interest and are recognized by autologous cytotoxic Tlymphocytes which then propagate.

A similar effect can be achieved by combining an antigen or an antigenpeptide with an adjuvant in order to make incorporation intoantigen-presenting cells in vivo possible. The antigen or antigenpeptide may be represented as protein, as DNA (e.g. within a vector) oras RNA. The antigen may be processed to produce a peptide partner forthe MHC molecule, while a fragment thereof may be presented without theneed for further processing. The latter is the case in particular, ifthese can bind to MHC molecules. Preference is given to administrationforms in which the complete antigen is processed in vivo by a dendriticcell, since this may also produce T helper cell responses which areneeded for an effective immune response (Ossendorp et al., Immunol Lett.(2000), 74:75-9; Ossendorp et al. (1998), J. Exp. Med. 187:693-702. Ingeneral, it is possible to administer an effective amount of thetumor-associated antigen to a patient by intradermal injection, forexample. However, injection may also be carried out intranodally into alymph node (Maloy et al. (2001), Proc Natl Acad Sci USA 98:3299-303).

According to the invention the term “artificial T cell receptor” issynonymous with the terms “chimeric T cell receptor” and “chimericantigen receptor (CAR)”.

These terms relate to engineered receptors, which confer an arbitraryspecificity such as the specificity of a monoclonal antibody onto animmune effector cell such as a T cell. In this way, a large number ofcancer-specific T cells can be generated for adoptive cell transfer.Thus, an artificial T cell receptor may be present on T cells, e.g.instead of or in addition to the T cell's own T cell receptor. Such Tcells do not necessarily require processing and presentation of anantigen for recognition of the target cell but rather may recognizepreferably with specificity any antigen present on a target cell.Preferably, said artificial T cell receptor is expressed on the surfaceof the cells. For the purpose of the present invention T cellscomprising an artificial T cell receptor are comprised by the term “Tcell” as used herein.

In one embodiment, a single-chain variable fragment (scFv) derived froma monoclonal antibody is fused to CD3-zeta transmembrane and endodomainSuch molecules result in the transmission of a zeta signal in responseto recognition by the scFv of its antigen target on a target cell andkilling of the target cell that expresses the target antigen. Antigenrecognition domains which also may be used include among others T-cellreceptor (TCR) alpha and beta single chains. In fact almost anythingthat binds a given target with high affinity can be used as an antigenrecognition domain.

Following antigen recognition, receptors cluster and a signal istransmitted to the cell. In this respect, a “T cell signaling domain” isa domain, preferably an endodomain, which transmits an activation signalto the T cell after antigen is bound. The most commonly used endodomaincomponent is CD3-zeta.

Adoptive cell transfer therapy with CAR-engineered T cells expressingchimeric antigen receptors is a promising anti-cancer therapeutic asCAR-modified T cells can be engineered to target virtually any tumorantigen. For example, patient's T cells may be genetically engineered toexpress CARs specifically directed towards antigens on the patient'stumor cells, then infused back into the patient.

According to the invention an artificial T cell receptor may replace thefunction of a T cell receptor as described above and, in particular, mayconfer reactivity such as cytolytic activity to a cell such as a T cellas described above. However, in contrast to the binding of the T cellreceptor to an antigen peptide-MHC complex as described above, anartificial T cell receptor may bind to an antigen, in particularexpressed on the cell surface.

The T-cell surface glycoprotein CD3-zeta chain is a protein that inhumans is encoded by the CD247 gene. CD3-zeta together with T-cellreceptor alpha/beta and gamma/delta heterodimers and CD3-gamma, -delta,and -epsilon, forms the T-cell receptor-CD3 complex. The zeta chainplays an important role in coupling antigen recognition to severalintracellular signal-transduction pathways. The term “CD3-zeta”preferably relates to human CD3-zeta, and, in particular, to a proteincomprising, preferably consisting of the amino acid sequence of SEQ IDNO: 45 of the sequence listing or a variant of said amino acid sequence.

CD28 (Cluster of Differentiation 28) is one of the molecules expressedon T cells that provide co-stimulatory signals, which are required for Tcell activation. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2).Stimulation through CD28 in addition to the T cell receptor (TCR) canprovide a potent co-stimulatory signal to T cells for the production ofvarious interleukins (IL-6 in particular). The term “CD28” preferablyrelates to human CD28, and, in particular, to a protein comprising,preferably consisting of the amino acid sequence of SEQ ID NO: 44 of thesequence listing or a variant of said amino acid sequence.

According to the invention, CARs may generally comprise three domains.

The first domain is the binding domain which recognizes and binds CLDN6.

The second domain is the co-stimulation domain. The co-stimulationdomain serves to enhance the proliferation and survival of the cytotoxiclymphocytes upon binding of the CAR to a targeted moiety. The identityof the co-stimulation domain is limited only in that it has the abilityto enhance cellular proliferation and survival upon binding of thetargeted moiety by the CAR. Suitable co-stimulation domains includeCD28, CD137 (4-1BB), a member of the tumor necrosis factor (TNF)receptor family, CD134 (OX40), a member of the TNFR-superfamily ofreceptors, and CD278 (ICOS), a CD28-superfamily co-stimulatory moleculeexpressed on activated T cells. The skilled person will understand thatsequence variants of these noted co-stimulation domains can be usedwithout adversely impacting the invention, where the variants have thesame or similar activity as the domain on which they are modeled. Suchvariants will have at least about 80% sequence identity to the aminoacid sequence of the domain from which they are derived. In someembodiments of the invention, the CAR constructs comprise twoco-stimulation domains. While the particular combinations include allpossible variations of the four noted domains, specific examples includeCD28+CD137 (4-1BB) and CD28+CD134 (OX40).

The third domain is the activation signaling domain (or T cell signalingdomain). The activation signaling domain serves to activate cytotoxiclymphocytes upon binding of the CAR to CLDN6. The identity of theactivation signaling domain is limited only in that it has the abilityto induce activation of the selected cytotoxic lymphocyte upon bindingof the CLDN6 by the CAR. Suitable activation signaling domains includethe T cell CD3[zeta] chain and Fc receptor [gamma]. The skilled artisanwill understand that sequence variants of these noted activationsignaling domains can be used without adversely impacting the invention,where the variants have the same or similar activity as the domain onwhich they are modeled. Such variants will have at least about 80%sequence identity to the amino acid sequence of the domain from whichthey are derived.

The CARs of the present invention may comprise the three domains,together in the form of a fusion protein. Such fusion proteins willgenerally comprise a binding domain, one or more co-stimulation domains,and an activation signaling domain, linked in a N-terminal to C-terminaldirection. However, the CARs of the present invention are not limited tothis arrangement and other arrangements are acceptable and include abinding domain, an activation signaling domain, and one or moreco-stimulation domains. It will be understood that because the bindingdomain must be free to bind CLDN6, the placement of the binding domainin the fusion protein will generally be such that display of the regionon the exterior of the cell is achieved. In the same manner, because theco-stimulation and activation signaling domains serve to induce activityand proliferation of the cytotoxic lymphocytes, the fusion protein willgenerally display these two domains in the interior of the cell. TheCARs may include additional elements, such as a signal peptide to ensureproper export of the fusion protein to the cells surface, atransmembrane domain to ensure the fusion protein is maintained as anintegral membrane protein, and a hinge domain (or spacer region) thatimparts flexibility to the binding domain and allows strong binding toCLDN6.

The cells used in connection with the CAR system of the presentinvention are preferably T cells, in particular cytotoxic lymphocytes,preferably selected from cytotoxic T cells, natural killer (NK) cells,and lymphokine-activated killer (LAK) cells. Upon activation, each ofthese cytotoxic lymphocytes triggers the destruction of target cells.For example, cytotoxic T cells trigger the destruction of target cellsby either or both of the following means. First, upon activation T cellsrelease cytotoxins such as perform, granzymes, and granulysin. Perforinand granulysin create pores in the target cell, and granzymes enter thecell and trigger a caspase cascade in the cytoplasm that inducesapoptosis (programmed cell death) of the cell. Second, apoptosis can beinduced via Fas-Fas ligand interaction between the T cells and targettumor cells. The cytotoxic lymphocytes will preferably be autologouscells, although heterologous cells or allogenic cells can be used.

According to the invention, a “reference” such as a reference sample orreference organism may be used to correlate and compare the resultsobtained in the methods of the invention from a test sample or testorganism. Typically the reference organism is a healthy organism, inparticular an organism which does not suffer from a disease such as acancer disease. A “reference value” or “reference level” can bedetermined from a reference empirically by measuring a sufficientlylarge number of references. Preferably the reference value is determinedby measuring at least 2, preferably at least 3, preferably at least 5,preferably at least 8, preferably at least 12, preferably at least 20,preferably at least 30, preferably at least 50, or preferably at least100 references.

According to the invention, the term “binding agent” includes anycompound that has a binding capacity to a target. Preferably, suchbinding agent comprises at least one binding domain for the target. Theterm includes molecules such as antibodies and antibody fragments,bispecific or multispecific molecules, chimeric antigen receptors (CARs)and all artificial binding molecules (scaffolds) having a bindingcapacity to the target including but not limited to nanobodies,affibodies, anticalins, DARPins, monobodies, avimers, and microbodies.In one embodiment said binding is a specific binding.

The term “immunoglobulin” relates to proteins of the immunoglobulinsuperfamily, preferably to antigen receptors such as antibodies or the Bcell receptor (BCR). The immunoglobulins are characterized by astructural domain, i.e., the immunoglobulin domain, having acharacteristic immunoglobulin (Ig) fold. The term encompasses membranebound immunoglobulins as well as soluble immunoglobulins. Membrane boundimmunoglobulins are also termed surface immunoglobulins or membraneimmunoglobulins, which are generally part of the BCR. Solubleimmunoglobulins are generally termed antibodies Immunoglobulinsgenerally comprise several chains, typically two identical heavy chainsand two identical light chains which are linked via disulfide bonds.These chains are primarily composed of immunoglobulin domains, such asthe V_(L) (variable light chain) domain, C_(L) (constant light chain)domain, and the C_(H) (constant heavy chain) domains C_(H)1, C_(H)2,C_(H)3, and C_(H)4. There are five types of mammalian immunoglobulinheavy chains, i.e., α, δ, ε, γ, and μ which account for the differentclasses of antibodies, i.e., IgA, IgD, IgE, IgG, and IgM. As opposed tothe heavy chains of soluble immunoglobulins, the heavy chains ofmembrane or surface immunoglobulins comprise a transmembrane domain anda short cytoplasmic domain at their carboxy-terminus. In mammals thereare two types of light chains, i.e., lambda and kappa. Theimmunoglobulin chains comprise a variable region and a constant region.The constant region is essentially conserved within the differentisotypes of the immunoglobulins, wherein the variable part is highlydivers and accounts for antigen recognition.

The term “antibody” refers to a glycoprotein comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. The term “antibody” includes monoclonal antibodies, recombinantantibodies, human antibodies, humanized antibodies and chimericantibodies. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region.Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The VH andVL regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

The term “monoclonal antibody” as used herein refers to a preparation ofantibody molecules of single molecular composition. A monoclonalantibody displays a single binding specificity and affinity. In oneembodiment, the monoclonal antibodies are produced by a hybridoma whichincludes a B cell obtained from a non-human animal, e g, mouse, fused toan immortalized cell.

The term “recombinant antibody”, as used herein, includes all antibodiesthat are prepared, expressed, created or isolated by recombinant means,such as (a) antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal with respect to the immunoglobulin genesor a hybridoma prepared therefrom, (b) antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial antibody library,and (d) antibodies prepared, expressed, created or isolated by any othermeans that involve splicing of immunoglobulin gene sequences to otherDNA sequences.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. Human antibodies may include aminoacid residues not encoded by human germline immunoglobulin sequences(e.g., mutations introduced by random or site-specific mutagenesis invitro or by somatic mutation in vivo).

The term “humanized antibody” refers to a molecule having an antigenbinding site that is substantially derived from an immunoglobulin from anon-human species, wherein the remaining immunoglobulin structure of themolecule is based upon the structure and/or sequence of a humanimmunoglobulin. The antigen binding site may either comprise completevariable domains fused onto constant domains or only the complementaritydetermining regions (CDR) grafted onto appropriate framework regions inthe variable domains. Antigen binding sites may be wild-type or modifiedby one or more amino acid substitutions, e.g. modified to resemble humanimmunoglobulins more closely. Some forms of humanized antibodiespreserve all CDR sequences (for example a humanized mouse antibody whichcontains all six CDRs from the mouse antibody). Other forms have one ormore CDRs which are altered with respect to the original antibody.

The term “chimeric antibody” refers to those antibodies wherein oneportion of each of the amino acid sequences of heavy and light chains ishomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular class, while theremaining segment of the chain is homologous to corresponding sequencesin another. Typically the variable region of both light and heavy chainsmimics the variable regions of antibodies derived from one species ofmammals, while the constant portions are homologous to sequences ofantibodies derived from another. One clear advantage to such chimericforms is that the variable region can conveniently be derived frompresently known sources using readily available B-cells or hybridomasfrom non-human host organisms in combination with constant regionsderived from, for example, human cell preparations. While the variableregion has the advantage of ease of preparation and the specificity isnot affected by the source, the constant region being human, is lesslikely to elicit an immune response from a human subject when theantibodies are injected than would the constant region from a non humansource. However the definition is not limited to this particularexample.

Antibodies may be derived from different species, including but notlimited to mouse, rat, rabbit, guinea pig and human.

Antibodies described herein include IgA such as IgA1 or IgA2, IgG1,IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments,the antibody is an IgG1 antibody, more particularly an IgG1, kappa orIgG1, lambda isotype (i.e. IgG1, κ, λ), an IgG2a antibody (e.g. IgG2a,κ, λ), an IgG2b antibody (e.g. IgG2b, κ, λ), an IgG3 antibody (e.g.IgG3, κ, λ) or an IgG4 antibody (e.g. IgG4, κ, λ).

The antibodies described herein are preferably isolated. An “isolatedantibody” as used herein, is intended to refer to an antibody which issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds toCLDN6 is substantially free of antibodies that specifically bindantigens other than CLDN6). An isolated antibody that specifically bindsto an epitope, isoform or variant of human CLDN6 may, however, havecross-reactivity to other related antigens, e.g., from other species(e.g., CLDN6 species homologs). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals. In oneembodiment of the invention, a combination of “isolated” monoclonalantibodies relates to antibodies having different specificities andbeing combined in a well defined composition or mixture.

The terms “antigen-binding portion” of an antibody (or simply “bindingportion”) or “antigen-binding fragment” of an antibody (or simply“binding fragment”) or similar terms refer to one or more fragments ofan antibody that retain the ability to specifically bind to an antigen.It has been shown that the antigen-binding function of an antibody canbe performed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) Fab fragments, monovalent fragments consisting ofthe VL, VH, CL and CH domains; (ii) F(ab′)₂ fragments, bivalentfragments comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) Fd fragments consisting of the VH and CHdomains; (iv) Fv fragments consisting of the VL and VH domains of asingle arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature341: 544-546), which consist of a VH domain; (vi) isolatedcomplementarity determining regions (CDR), and (vii) combinations of twoor more isolated CDRs which may optionally be joined by a syntheticlinker. Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding fragment” ofan antibody. A further example is binding-domain immunoglobulin fusionproteins comprising (i) a binding domain polypeptide that is fused to animmunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavychain CH2 constant region fused to the hinge region, and (iii) animmunoglobulin heavy chain CH3 constant region fused to the CH2 constantregion. The binding domain polypeptide can be a heavy chain variableregion or a light chain variable region. The binding-domainimmunoglobulin fusion proteins are further disclosed in US 2003/0118592and US 2003/0133939. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

According to the invention, the term “binding domain for CLDN6” includesand preferably relates to the antigen-binding portion of a CLDN6antibody, i.e. an antibody which is directed against CLDN6 and ispreferably specific for CLDN6.

The term “binding domain” characterizes in connection with the presentinvention a structure, e.g. of an antibody, which binds to/interactswith a given target structure/antigen/epitope. Thus, the binding domainaccording to the invention designates an “antigen-interaction-site”.

All antibodies and derivatives of antibodies such as antibody fragmentsas described herein for the purposes of the invention are encompassed bythe term “antibody”.

Antibodies can be produced by a variety of techniques, includingconventional monoclonal antibody methodology, e.g., the standard somaticcell hybridization technique of Kohler and Milstein, Nature 256: 495(1975). Although somatic cell hybridization procedures are preferred, inprinciple, other techniques for producing monoclonal antibodies can beemployed, e.g., viral or oncogenic transformation of B-lymphocytes orphage display techniques using libraries of antibody genes.

The preferred animal system for preparing hybridomas that secretemonoclonal antibodies is the murine system. Hybridoma production in themouse is a very well established procedure. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. Fusion partners (e.g., murine myeloma cells) and fusionprocedures are also known.

Other preferred animal systems for preparing hybridomas that secretemonoclonal antibodies are the rat and the rabbit system (e.g. describedin Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995),see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)).

To generate antibodies, mice can be immunized with carrier-conjugatedpeptides derived from the antigen sequence, i.e. the sequence againstwhich the antibodies are to be directed, an enriched preparation ofrecombinantly expressed antigen or fragments thereof and/or cellsexpressing the antigen, as described. Alternatively, mice can beimmunized with DNA encoding the antigen or fragments thereof. In theevent that immunizations using a purified or enriched preparation of theantigen do not result in antibodies, mice can also be immunized withcells expressing the antigen, e.g., a cell line, to promote immuneresponses.

The immune response can be monitored over the course of the immunizationprotocol with plasma and serum samples being obtained by tail vein orretroorbital bleeds. Mice with sufficient titers of immunoglobulin canbe used for fusions. Mice can be boosted intraperitonealy orintravenously with antigen expressing cells 3 days before sacrifice andremoval of the spleen to increase the rate of specific antibodysecreting hybridomas.

To generate hybridomas producing monoclonal antibodies, splenocytes andlymph node cells from immunized mice can be isolated and fused to anappropriate immortalized cell line, such as a mouse myeloma cell line.The resulting hybridomas can then be screened for the production ofantigen-specific antibodies. Individual wells can then be screened byELISA for antibody secreting hybridomas. By Immunofluorescence and FACSanalysis using antigen expressing cells, antibodies with specificity forthe antigen can be identified. The antibody secreting hybridomas can bereplated, screened again, and if still positive for monoclonalantibodies can be subcloned by limiting dilution. The stable subclonescan then be cultured in vitro to generate antibody in tissue culturemedium for characterization.

The ability of antibodies and other binding agents to bind an antigencan be determined using standard binding assays (e.g., ELISA, WesternBlot, Immunofluorescence and flow cytometric analysis).

Antibodies and derivatives of antibodies are useful for providingbinding domains such as antibody fragments, in particular for providingVL and VH regions.

A binding domain for CLDN6 which may be present within an artificial Tcell receptor has the ability of binding to CLDN6, i.e. the ability ofbinding to an epitope present in CLDN6, preferably an epitope locatedwithin the extracellular domains of CLDN6, in particular the firstextracellular loop, preferably amino acid positions 28 to 76 of CLDN6 orthe second extracellular loop, preferably amino acid positions 141 to159 of CLDN6. In particular embodiments, a binding domain for CLDN6binds to an epitope on CLDN6 which is not present on CLDN9. Preferably,a binding domain for CLDN6 binds to an epitope on CLDN6 which is notpresent on CLDN4 and/or CLDN3. Most preferably, a binding domain forCLDN6 binds to an epitope on CLDN6 which is not present on a CLDNprotein other than CLDN6.

A binding domain for CLDN6 preferably binds to CLDN6 but not to CLDN9and preferably does not bind to CLDN4 and/or CLDN3. Preferably, abinding domain for CLDN6 is specific for CLDN6. Preferably, a bindingdomain for CLDN6 binds to CLDN6 expressed on the cell surface. Inparticular preferred embodiments, a binding domain for CLDN6 binds tonative epitopes of CLDN6 present on the surface of living cells.

In a preferred embodiment, a binding domain for CLDN6 comprises a heavychain variable region (VH) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 30, 32, 34 and 36 or a fragmentthereof, or a variant of said amino acid sequence or fragment.

In a preferred embodiment, a binding domain for CLDN6 comprises a lightchain variable region (VL) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 31, 33, 35, 37, 38 and 39 or afragment thereof, or a variant of said amino acid sequence or fragment.

In certain preferred embodiments, a binding domain for CLDN6 comprises acombination of heavy chain variable region (VH) and light chain variableregion (VL) selected from the following possibilities (i) to (xi):

(i) the VH comprises an amino acid sequence represented by SEQ ID NO: 30or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 31 or a fragment thereof,

(ii) the VH comprises an amino acid sequence represented by SEQ ID NO:32 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 33 or a fragment thereof,

(iii) the VH comprises an amino acid sequence represented by SEQ ID NO:34 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 35 or a fragment thereof,

(iv) the VH comprises an amino acid sequence represented by SEQ ID NO:36 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 37 or a fragment thereof,

(v) the VH comprises an amino acid sequence represented by SEQ ID NO: 32or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 31 or a fragment thereof,

(vi) the VH comprises an amino acid sequence represented by SEQ ID NO:32 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 38 or a fragment thereof,

(vii) the VH comprises an amino acid sequence represented by SEQ ID NO:32 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 39 or a fragment thereof.

In a particularly preferred embodiment, a binding domain for CLDN6comprises the following combination of heavy chain variable region (VH)and light chain variable region (VL): the VH comprises an amino acidsequence represented by SEQ ID NO: 32 or a fragment thereof and the VLcomprises an amino acid sequence represented by SEQ ID NO: 39 or afragment thereof.

The term “fragment” refers, in particular, to one or more of thecomplementarity-determining regions (CDRs), preferably at least the CDR3variable region, of the heavy chain variable region (VH) and/or of thelight chain variable region (VL). In one embodiment said one or more ofthe complementarity-determining regions (CDRs) are selected from a setof complementarity-determining regions CDR1, CDR2 and CDR3. In aparticularly preferred embodiment, the term “fragment” refers to thecomplementarity-determining regions CDR1, CDR2 and CDR3 of the heavychain variable region (VH) and/or of the light chain variable region(VL).

In one embodiment a binding domain for CLDN6 comprising one or moreCDRs, a set of CDRs or a combination of sets of CDRs as described hereincomprises said CDRs together with their intervening framework regions.Preferably, the portion will also include at least about 50% of eitheror both of the first and fourth framework regions, the 50% being theC-terminal 50% of the first framework region and the N-terminal 50% ofthe fourth framework region. Construction of binding domains made byrecombinant DNA techniques may result in the introduction of residues N-or C-terminal to the variable regions encoded by linkers introduced tofacilitate cloning or other manipulation steps, including theintroduction of linkers to join variable regions of the invention tofurther protein sequences including immunoglobulin heavy chains, othervariable domains (for example in the production of diabodies) or proteinlabels.

In one embodiment a binding domain comprising one or more CDRs, a set ofCDRs or a combination of sets of CDRs as described herein comprises saidCDRs in a human antibody framework.

The term “binding” according to the invention preferably relates to aspecific binding.

According to the present invention, an agent such as a T cell receptoror an antibody is capable of binding to a predetermined target if it hasa significant affinity for said predetermined target and binds to saidpredetermined target in standard assays. “Affinity” or “bindingaffinity” is often measured by equilibrium dissociation constant(K_(D)). Preferably, the term “significant affinity” refers to thebinding to a predetermined target with a dissociation constant (K_(D))of 10⁻⁵ M or lower, 10⁻⁶ M or lower, 10⁻⁷ M or lower, 10⁻⁸M or lower,10⁻⁹M or lower, 10⁻¹⁰M or lower, 10⁻¹¹ M or lower, or 10⁻¹²M or lower.

An agent is not (substantially) capable of binding to a target if it hasno significant affinity for said target and does not bind significantly,in particular does not bind detectably, to said target in standardassays. Preferably, the agent does not detectably bind to said target ifpresent in a concentration of up to 2, preferably 10, more preferably20, in particular 50 or 100 μg/ml or higher. Preferably, an agent has nosignificant affinity for a target if it binds to said target with aK_(D) that is at least 10-fold, 100-fold, 10³-fold, 10⁴-fold, 10⁵-fold,or 10⁶-fold higher than the K_(D) for binding to the predeterminedtarget to which the agent is capable of binding. For example, if theK_(D) for binding of an agent to the target to which the agent iscapable of binding is 10⁻⁷ M, the K_(D) for binding to a target forwhich the agent has no significant affinity would be at least 10⁻⁶ M,10⁻⁵ M, 10⁻⁴ M, 10⁻³ M, 10⁻² M, or 10⁻¹ M.

An agent is specific for a predetermined target if it is capable ofbinding to said predetermined target while it is not (substantially)capable of binding to other targets, i.e. has no significant affinityfor other targets and does not significantly bind to other targets instandard assays. According to the invention, an agent is specific forCLDN6 if it is capable of binding to CLDN6 but is not (substantially)capable of binding to other targets. Preferably, an agent is specificfor CLDN6 if the affinity for and the binding to such other targets doesnot significantly exceed the affinity for or binding to CLDN6-unrelatedproteins such as bovine serum albumin (BSA), casein, human serum albumin(HSA) or non-claudin transmembrane proteins such as MHC molecules ortransferrin receptor or any other specified polypeptide. Preferably, anagent is specific for a predetermined target if it binds to said targetwith a K_(D) that is at least 10-fold, 100-fold, 10³-fold, 10⁴-fold,10⁵-fold, or 10⁶-fold lower than the K_(D) for binding to a target forwhich it is not specific. For example, if the K_(D) for binding of anagent to the target for which it is specific is 10⁻⁷ M, the K_(D) forbinding to a target for which it is not specific would be at least 10⁻⁶M, 10⁻⁵ M, 10⁻⁴ M, 10⁻³ M, 10⁻² M, or 10⁻¹ M.

Binding of an agent to a target can be determined experimentally usingany suitable method; see, for example, Berzofsky et al.,“Antibody-Antigen Interactions” In Fundamental Immunology, Paul, W. E.,Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H.Freeman and Company New York, N Y (1992), and methods described herein.Affinities may be readily determined using conventional techniques, suchas by equilibrium dialysis; by using the BIAcore 2000 instrument, usinggeneral procedures outlined by the manufacturer; by radioimmunoassayusing radiolabeled target antigen; or by another method known to theskilled artisan. The affinity data may be analyzed, for example, by themethod of Scatchard et al., Ann N.Y. Acad. ScL, 51:660 (1949). Themeasured affinity of a particular antibody-antigen interaction can varyif measured under different conditions, e.g., salt concentration, pH.Thus, measurements of affinity and other antigen-binding parameters,e.g., K_(D), IC₅₀, are preferably made with standardized solutions ofantibody and antigen, and a standardized buffer.

It is to be understood that the peptide and protein agents describedherein may be provided in vitro or in vivo in the form of a nucleic acidsuch as RNA encoding the agent and/or in the form of a host cellcomprising a nucleic acid such as RNA encoding the agent. In particular,a variety of methods may be used to introduce CAR constructs into Tcells including non-viral-based DNA transfection, transposon-basedsystems and viral-based systems. Non-viral-based DNA transfection haslow risk of insertional mutagenesis. Transposon-based systems canintegrate transgenes more efficiently than plasmids that do not containan integrating element. Viral-based systems include the use ofγ-retroviruses and lentiviral vectors. γ-Retroviruses are relativelyeasy to produce, efficiently and permanently transduce T cells, and havepreliminarily proven safe from an integration standpoint in primaryhuman T cells. Lentiviral vectors also efficiently and permanentlytransduce T cells but are more expensive to manufacture. They are alsopotentially safer than retrovirus based systems.

The peptide and protein agents described herein may be delivered to apatient by administering a nucleic acid such as RNA encoding the agentand/or by administering a host cell comprising a nucleic acid such asRNA encoding the agent. A nucleic acid when administered to a patientmay be present in naked form or in a suitable delivery vehicle such asin the form of liposomes or viral particles, or within a host cell. Thenucleic acid provided can produce the agent over extended time periodsin a sustained manner mitigating the instability at least partiallyobserved for therapeutic proteins. If a nucleic acid is administered toa patient without being present within a host cell, it is preferablytaken up by cells of the patient for expression of the agent encoded bythe nucleic acid. If a nucleic acid is administered to a patient whilebeing present within a host cell, it is preferably expressed by the hostcell within the patient so as to produce the agent encoded by thenucleic acid.

The term “nucleic acid”, as used herein, is intended to include DNA andRNA such as genomic DNA, cDNA, mRNA, recombinantly produced andchemically synthesized molecules. A nucleic acid may be single-strandedor double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) orsynthetic RNA. According to the invention, a nucleic acid is preferablyan isolated nucleic acid.

Nucleic acids may be comprised in a vector. The term “vector” as usedherein includes any vectors known to the skilled person includingplasmid vectors, cosmid vectors, phage vectors such as lambda phage,viral vectors such as adenoviral or baculoviral vectors, or artificialchromosome vectors such as bacterial artificial chromosomes (BAC), yeastartificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Saidvectors include expression as well as cloning vectors. Expressionvectors comprise plasmids as well as viral vectors and generally containa desired coding sequence and appropriate DNA sequences necessary forthe expression of the operably linked coding sequence in a particularhost organism (e.g., bacteria, yeast, plant, insect, or mammal) or in invitro expression systems. Cloning vectors are generally used to engineerand amplify a certain desired DNA fragment and may lack functionalsequences needed for expression of the desired DNA fragments.

In the context of the present invention, the term “RNA” relates to amolecule which comprises ribonucleotide residues and preferably beingentirely or substantially composed of ribonucleotide residues.“Ribonucleotide” relates to a nucleotide with a hydroxyl group at the2′-position of a (3-D-ribofuranosyl group. The term includes doublestranded RNA, single stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as modified RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of a RNA or internally,for example at one or more nucleotides of the RNA. Nucleotides in RNAmolecules can also comprise non-standard nucleotides, such asnon-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs or analogs of naturally-occurring RNA.

According to the present invention, the term “RNA” includes andpreferably relates to “mRNA” which means “messenger RNA” and relates toa “transcript” which may be produced using DNA as template and encodes apeptide or protein. mRNA typically comprises a 5′ non translated region(5′-UTR), a protein or peptide coding region and a 3′ non translatedregion (3′-UTR). mRNA has a limited halftime in cells and in vitro.Preferably, mRNA is produced by in vitro transcription using a DNAtemplate. In one embodiment of the invention, the RNA is obtained by invitro transcription or chemical synthesis. The in vitro transcriptionmethodology is known to the skilled person. For example, there is avariety of in vitro transcription kits commercially available.

In one embodiment of the present invention, RNA is self-replicating RNA,such as single stranded self-replicating RNA. In one embodiment, theself-replicating RNA is single stranded RNA of positive sense. In oneembodiment, the self-replicating RNA is viral RNA or RNA derived fromviral RNA. In one embodiment, the self-replicating RNA is alphaviralgenomic RNA or is derived from alphaviral genomic RNA. In oneembodiment, the self-replicating RNA is a viral gene expression vector.In one embodiment, the virus is Semliki forest virus. In one embodiment,the self-replicating RNA contains one or more transgenes at least one ofsaid transgenes encoding the agents described herein. In one embodiment,if the RNA is viral RNA or derived from viral RNA, the transgenes maypartially or completely replace viral sequences such as viral sequencesencoding structural proteins. In one embodiment, the self-replicatingRNA is in vitro transcribed RNA.

In order to increase expression and/or stability of the RNA usedaccording to the present invention, it may be modified, preferablywithout altering the sequence of the expressed peptide or protein.

The term “modification” in the context of RNA as used according to thepresent invention includes any modification of RNA which is notnaturally present in said RNA.

In one embodiment of the invention, the RNA used according to theinvention does not have uncapped 5′-triphosphates. Removal of suchuncapped 5′-triphosphates can be achieved by treating RNA with aphosphatase.

The RNA according to the invention may have modified naturally occurringor synthetic ribonucleotides in order to increase its stability and/ordecrease cytotoxicity. For example, in one embodiment, in the RNA usedaccording to the invention 5-methylcytidine is substituted partially orcompletely, preferably completely, for cytidine. Alternatively oradditionally, in one embodiment, in the RNA used according to theinvention pseudouridine is substituted partially or completely,preferably completely, for uridine.

In one embodiment, the term “modification” relates to providing an RNAwith a 5′-cap or 5′-cap analog. The term “5′-cap” refers to a capstructure found on the 5′-end of an mRNA molecule and generally consistsof a guanosine nucleotide connected to the mRNA via an unusual 5′ to 5′triphosphate linkage. In one embodiment, this guanosine is methylated atthe 7-position. The term “conventional 5′-cap” refers to a naturallyoccurring RNA 5′-cap, preferably to the 7-methylguanosine cap (m7G). Inthe context of the present invention, the term “5′-cap” includes a5′-cap analog that resembles the RNA cap structure and is modified topossess the ability to stabilize RNA if attached thereto, preferably invivo and/or in a cell.

Providing an RNA with a 5′-cap or 5′-cap analog may be achieved by invitro transcription of a DNA template in the presence of said 5′-cap or5′-cap analog, wherein said 5′-cap is co-transcriptionally incorporatedinto the generated RNA strand, or the RNA may be generated, for example,by in vitro transcription, and the 5′-cap may be attached to the RNApost-transcriptionally using capping enzymes, for example, cappingenzymes of vaccinia virus.

The RNA may comprise further modifications. For example, a furthermodification of the RNA used in the present invention may be anextension or truncation of the naturally occurring poly(A) tail or analteration of the 5′- or 3′-untranslated regions (UTR) such asintroduction of a UTR which is not related to the coding region of saidRNA, for example, the insertion of one or more, preferably two copies ofa 3′-UTR derived from a globin gene, such as alpha2-globin,alpha1-globin, beta-globin, preferably beta-globin, more preferablyhuman beta-globin.

Therefore, in order to increase stability and/or expression of the RNAused according to the present invention, it may be modified so as to bepresent in conjunction with a poly-A sequence, preferably having alength of 10 to 500, more preferably 30 to 300, even more preferably 65to 200 and especially 100 to 150 adenosine residues. In an especiallypreferred embodiment the poly-A sequence has a length of approximately120 adenosine residues. In addition, incorporation of two or more 3′-nontranslated regions (UTR) into the 3′-non translated region of an RNAmolecule can result in an enhancement in translation efficiency. In oneparticular embodiment the 3′-UTR is derived from the human β-globingene.

The term “stability” of RNA relates to the “half-life” of RNA.“Half-life” relates to the period of time which is needed to eliminatehalf of the activity, amount, or number of molecules. In the context ofthe present invention, the half-life of an RNA is indicative for thestability of said RNA. The half-life of RNA may influence the “durationof expression” of the RNA. It can be expected that RNA having a longhalf-life will be expressed for an extended time period.

In the context of the present invention, the term “transcription”relates to a process, wherein the genetic code in a DNA sequence istranscribed into RNA. Subsequently, the RNA may be translated intoprotein. According to the present invention, the term “transcription”comprises “in vitro transcription”, wherein the term “in vitrotranscription” relates to a process wherein RNA, in particular mRNA, isin vitro synthesized in a cell-free system, preferably using appropriatecell extracts. Preferably, cloning vectors are applied for thegeneration of transcripts. These cloning vectors are generallydesignated as transcription vectors and are according to the presentinvention encompassed by the term “vector”.

The term “translation” according to the invention relates to the processin the ribosomes of a cell by which a strand of messenger RNA directsthe assembly of a sequence of amino acids to make a peptide or protein.

Nucleic acids may, according to the invention, be present alone or incombination with other nucleic acids, which may be homologous orheterologous. In preferred embodiments, a nucleic acid is functionallylinked to expression control sequences which may be homologous orheterologous with respect to said nucleic acid. The term “homologous”means that the nucleic acids are also functionally linked naturally andthe term “heterologous” means that the nucleic acids are notfunctionally linked naturally.

A nucleic acid and an expression control sequence are “functionally”linked to one another, if they are covalently linked to one another insuch a way that expression or transcription of said nucleic acid isunder the control or under the influence of said expression controlsequence. If the nucleic acid is to be translated into a functionalprotein, then, with an expression control sequence functionally linkedto a coding sequence, induction of said expression control sequenceresults in transcription of said nucleic acid, without causing a frameshift in the coding sequence or said coding sequence not being capableof being translated into the desired protein or peptide.

The term “expression control sequence” or “expression control element”comprises according to the invention promoters, ribosome binding sites,enhancers and other control elements which regulate transcription of agene or translation of a mRNA. In particular embodiments of theinvention, the expression control sequences can be regulated. The exactstructure of expression control sequences may vary as a function of thespecies or cell type, but generally comprises 5′-untranscribed and 5′-and 3′-untranslated sequences which are involved in initiation oftranscription and translation, respectively, such as TATA box, cappingsequence, CAAT sequence, and the like. More specifically,5′-untranscribed expression control sequences comprise a promoter regionwhich includes a promoter sequence for transcriptional control of thefunctionally linked nucleic acid. Expression control sequences may alsocomprise enhancer sequences or upstream activator sequences.

The term “expression” is used according to the invention in its mostgeneral meaning and comprises the production of RNA and/or peptides orproteins, e.g. by transcription and/or translation. With respect to RNA,the term “expression” or “translation” relates in particular to theproduction of peptides or proteins. It also comprises partial expressionof nucleic acids. Moreover, expression can be transient or stable.According to the invention, the term expression also includes an“aberrant expression” or “abnormal expression”.

“Aberrant expression” or “abnormal expression” means according to theinvention that expression is altered, preferably increased, compared toa reference, e.g. a state in a subject not having a disease associatedwith aberrant or abnormal expression of a certain protein, e.g., a tumorantigen. An increase in expression refers to an increase by at least10%, in particular at least 20%, at least 50% or at least 100%, or more.In one embodiment, expression is only found in a diseased tissue, whileexpression in a healthy tissue is repressed.

The term “specifically expressed” means that a protein is essentiallyonly expressed in a specific tissue or organ. For example, a tumorantigen specifically expressed in gastric mucosa means that said proteinis primarily expressed in gastric mucosa and is not expressed in othertissues or is not expressed to a significant extent in other tissue ororgan types. Thus, a protein that is exclusively expressed in cells ofthe gastric mucosa and to a significantly lesser extent in any othertissue, such as testis, is specifically expressed in cells of thegastric mucosa. In some embodiments, a tumor antigen may also bespecifically expressed under normal conditions in more than one tissuetype or organ, such as in 2 or 3 tissue types or organs, but preferablyin not more than 3 different tissue or organ types. In this case, thetumor antigen is then specifically expressed in these organs. Forexample, if a tumor antigen is expressed under normal conditionspreferably to an approximately equal extent in lung and stomach, saidtumor antigen is specifically expressed in lung and stomach.

According to the invention, the term “nucleic acid encoding” means thatnucleic acid, if present in the appropriate environment, preferablywithin a cell, can be expressed to produce a protein or peptide itencodes.

Some aspects of the invention rely on the adoptive transfer of hostcells which are transfected in vitro with a nucleic acid such as RNAencoding an agent described herein and transferred to recipients such aspatients, preferably after ex vivo expansion from low precursorfrequencies to clinically relevant cell numbers. The host cells used fortreatment according to the invention may be autologous, allogeneic, orsyngeneic to a treated recipient.

The term “autologous” is used to describe anything that is derived fromthe same subject. For example, “autologous transplant” refers to atransplant of tissue or organs derived from the same subject. Suchprocedures are advantageous because they overcome the immunologicalbarrier which otherwise results in rejection.

The term “allogeneic” is used to describe anything that is derived fromdifferent individuals of the same species. Two or more individuals aresaid to be allogeneic to one another when the genes at one or more lociare not identical.

The term “syngeneic” is used to describe anything that is derived fromindividuals or tissues having identical genotypes, i.e., identical twinsor animals of the same inbred strain, or their tissues.

The term “heterologous” is used to describe something consisting ofmultiple different elements. As an example, the transfer of oneindividual's bone marrow into a different individual constitutes aheterologous transplant. A heterologous gene is a gene derived from asource other than the subject.

The term “transfection” relates to the introduction of nucleic acids, inparticular RNA, into a cell. For purposes of the present invention, theterm “transfection” also includes the introduction of a nucleic acidinto a cell or the uptake of a nucleic acid by such cell, wherein thecell may be present in a subject, e.g., a patient. Thus, according tothe present invention, a cell for transfection of a nucleic aciddescribed herein can be present in vitro or in vivo, e.g. the cell canform part of an organ, a tissue and/or an organism of a patient.According to the invention, transfection can be transient or stable. Forsome applications of transfection, it is sufficient if the transfectedgenetic material is only transiently expressed. Since the nucleic acidintroduced in the transfection process is usually not integrated intothe nuclear genome, the foreign nucleic acid will be diluted throughmitosis or degraded. Cells allowing episomal amplification of nucleicacids greatly reduce the rate of dilution. If it is desired that thetransfected nucleic acid actually remains in the genome of the cell andits daughter cells, a stable transfection must occur. RNA can betransfected into cells to transiently express its coded protein.

According to the present invention, any technique useful forintroducing, i.e. transferring or transfecting, nucleic acids into cellsmay be used. Preferably, RNA is transfected into cells by standardtechniques. Such techniques include electroporation, lipofection andmicroinjection. In one particularly preferred embodiment of the presentinvention, RNA is introduced into cells by electroporation.

Electroporation or electropermeabilization relates to a significantincrease in the electrical conductivity and permeability of the cellplasma membrane caused by an externally applied electrical field. It isusually used in molecular biology as a way of introducing some substanceinto a cell.

According to the invention it is preferred that introduction of nucleicacid encoding a protein or peptide into cells results in expression ofsaid protein or peptide.

The term “peptide” according to the invention comprises oligo- andpolypeptides and refers to substances comprising two or more, preferably3 or more, preferably 4 or more, preferably 6 or more, preferably 8 ormore, preferably 9 or more, preferably 10 or more, preferably 13 ormore, preferably 16 more, preferably 21 or more and up to preferably 8,10, 20, 30, 40 or 50, in particular 100 amino acids joined covalently bypeptide bonds. The term “protein” refers to large peptides, preferablyto peptides with more than 100 amino acid residues, but in general theterms “peptides” and “proteins” are synonyms and are usedinterchangeably herein.

According to the invention, a peptide may include natural amino acidsand non-natural amino acids. In one embodiment, a peptide merelyincludes natural amino acids.

According to the invention, the term “non-natural amino acid” refers toan amino acid having a structure different from those of the 20 naturalamino acid species. Since non-natural amino acids have structuressimilar to those of natural amino acids, non-natural amino acids may beclassified as derivatives or analogs of given natural amino acids.

Preferably, the proteins and peptides described according to theinvention have been isolated. The terms “isolated protein” or “isolatedpeptide” mean that the protein or peptide has been separated from itsnatural environment. An isolated protein or peptide may be in anessentially purified state. The term “essentially purified” means thatthe protein or peptide is essentially free of other substances withwhich it is associated in nature or in vivo.

The teaching given herein with respect to specific amino acid sequences,e.g. those shown in the sequence listing, is to be construed so as toalso relate to variants of said specific sequences resulting insequences which are functionally equivalent to said specific sequences,e.g. amino acid sequences exhibiting properties identical or similar tothose of the specific amino acid sequences. One important property is toretain binding of a peptide to an MHC molecule and/or to a T cellreceptor or of a T cell receptor to its target or to sustain effectorfunctions of a T cell. Preferably, a sequence modified with respect to aspecific sequence, when it replaces the specific sequence in a T cellreceptor retains binding of said T cell receptor to the target andpreferably functions of said T cell receptor or T cell carrying the Tcell receptor as described herein.

For example, the sequences shown in the sequence listing can be modifiedso as to remove one or more, preferably all free cysteine residues, inparticular by replacing the cysteine residues by amino acids other thancysteine, preferably serine, alanine, threonine, glycine, tyrosine,leucine or methionine, most preferably alanine or serine. For example,the cysteine at position 45 of the sequence shown in SEQ ID NO: 33 ofthe sequence listing or the corresponding cysteine in a sequencecomprising said sequence may be modified in this way.

It will be appreciated by those skilled in the art that in particularthe sequences of the CDR sequences, hypervariable and variable regionscan be modified without losing the ability to bind to a target. Forexample, CDR regions will be either identical or highly homologous tothe regions of antibodies specified herein. By “highly homologous” it iscontemplated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or1 or 2 substitutions may be made in the CDRs. In addition, thehypervariable and variable regions may be modified so that they showsubstantial homology with the regions specifically disclosed herein.

A peptide “variant” may retain the immunogenicity of a given peptide(e.g. the ability of the variant to react with T cell lines or clones isnot substantially diminished relative to the given peptide). In otherwords, the ability of a variant to react with T cell lines or clones maybe enhanced or unchanged, relative to the given peptide, or may bediminished by less than 50%, and preferably less than 20%, relative tothe given peptide.

A variant may be identified by evaluating its ability to bind to a MHCmolecule. In one preferred embodiment, a variant peptide has amodification such that the ability of the variant peptide to bind to aMHC molecule is increased relative to the given peptide. The ability ofthe variant peptide to bind to a MHC molecule may be increased by atleast 2-fold, preferably at least 3-fold, 4-fold, or 5-fold relative tothat of a given peptide. Accordingly, within certain preferredembodiments, a peptide comprises a variant in which 1 to 3 amino acidresides within an immunogenic portion are substituted such that theability to react with T cell lines or clones is statistically greaterthan that for the unmodified peptide. Such substitutions are preferablylocated within an MHC binding site of the peptide. Preferredsubstitutions allow increased binding to MHC class I or class IImolecules. Certain variants contain conservative substitutions.

The term “variant” according to the invention also includes mutants,splice variants, conformations, isoforms, allelic variants, speciesvariants and species homologs, in particular those which are naturallypresent. An allelic variant relates to an alteration in the normalsequence of a gene, the significance of which is often unclear. Completegene sequencing often identifies numerous allelic variants for a givengene. A species homolog is a nucleic acid or amino acid sequence with adifferent species of origin from that of a given nucleic acid or aminoacid sequence. The term “variant” shall encompass anyposttranslationally modified variants and conformation variants.

For the purposes of the present invention, “variants” of an amino acidsequence comprise amino acid insertion variants, amino acid additionvariants, amino acid deletion variants and/or amino acid substitutionvariants Amino acid deletion variants that comprise the deletion at theN-terminal and/or C-terminal end of the protein are also calledN-terminal and/or C-terminal truncation variants.

Amino acid insertion variants comprise insertions of single or two ormore amino acids in a particular amino acid sequence. In the case ofamino acid sequence variants having an insertion, one or more amino acidresidues are inserted into a particular site in an amino acid sequence,although random insertion with appropriate screening of the resultingproduct is also possible.

Amino acid addition variants comprise amino- and/or carboxy-terminalfusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50,or more amino acids.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence, such as by removal of 1, 2, 3, 5,10, 20, 30, 50, or more amino acids. The deletions may be in anyposition of the protein.

Amino acid substitution variants are characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place. Preference is given to the modifications being inpositions in the amino acid sequence which are not conserved betweenhomologous proteins or peptides and/or to replacing amino acids withother ones having similar properties. Preferably, amino acid changes inprotein variants are conservative amino acid changes, i.e.,substitutions of similarly charged or uncharged amino acids. Aconservative amino acid change involves substitution of one of a familyof amino acids which are related in their side chains. Naturallyoccurring amino acids are generally divided into four families acidic(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), and uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine) amino acids.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids.

Preferably the degree of similarity, preferably identity between a givenamino acid sequence and an amino acid sequence which is a variant ofsaid given amino acid sequence will be at least about 60%, 65%, 70%,80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity oridentity is given preferably for an amino acid region which is at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90% or about 100% of the entire length of thereference amino acid sequence. For example, if the reference amino acidsequence consists of 200 amino acids, the degree of similarity oridentity is given preferably for at least about 20, at least about 40,at least about 60, at least about 80, at least about 100, at least about120, at least about 140, at least about 160, at least about 180, orabout 200 amino acids, preferably continuous amino acids. In preferredembodiments, the degree of similarity or identity is given for theentire length of the reference amino acid sequence. The alignment fordetermining sequence similarity, preferably sequence identity can bedone with art known tools, preferably using the best sequence alignment,for example, using Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two amino acid sequencesindicates the percentage of amino acids that are identical between thesequences.

The term “percentage identity” is intended to denote a percentage ofamino acid residues which are identical between the two sequences to becompared, obtained after the best alignment, this percentage beingpurely statistical and the differences between the two sequences beingdistributed randomly and over their entire length. Sequence comparisonsbetween two amino acid sequences are conventionally carried out bycomparing these sequences after having aligned them optimally, saidcomparison being carried out by segment or by “window of comparison” inorder to identify and compare local regions of sequence similarity. Theoptimal alignment of the sequences for comparison may be produced,besides manually, by means of the local homology algorithm of Smith andWaterman, 1981, Ads App. Math. 2, 482, by means of the local homologyalgorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by meansof the similarity search method of Pearson and Lipman, 1988, Proc. NatlAcad. Sci. USA 85, 2444, or by means of computer programs which usethese algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA inWisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

Homologous amino acid sequences exhibit according to the invention atleast 40%, in particular at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% and preferably at least 95%, at least 98 or atleast 99% identity of the amino acid residues.

The amino acid sequence variants described herein may readily beprepared by the skilled person, for example, by recombinant DNAmanipulation. The manipulation of DNA sequences for preparing proteinsand peptides having substitutions, additions, insertions or deletions,is described in detail in Sambrook et al. (1989), for example.Furthermore, the peptides and amino acid variants described herein maybe readily prepared with the aid of known peptide synthesis techniquessuch as, for example, by solid phase synthesis and similar methods.

The invention includes derivatives of the peptides or proteins describedherein which are comprised by the terms “peptide” and “protein”.According to the invention, “derivatives” of proteins and peptides aremodified forms of proteins and peptides. Such modifications include anychemical modification and comprise single or multiple substitutions,deletions and/or additions of any molecules associated with the proteinor peptide, such as carbohydrates, lipids and/or proteins or peptides.In one embodiment, “derivatives” of proteins or peptides include thosemodified analogs resulting from glycosylation, acetylation,phosphorylation, amidation, palmitoylation, myristoylation,isoprenylation, lipidation, alkylation, derivatization, introduction ofprotective/blocking groups, proteolytic cleavage or binding to anantibody or to another cellular ligand. The term “derivative” alsoextends to all functional chemical equivalents of said proteins andpeptides. Preferably, a modified peptide has increased stability and/orincreased immunogenicity.

Also included are mimetics of peptides. Such mimetics may comprise aminoacids linked to one or more amino acid mimetics (i e., one or more aminoacids within the peptide may be replaced by an amino acid mimetic) ormay be entirely nonpeptide mimetics. An amino acid mimetic is a compoundthat is conformationally similar to an amino acid, e.g. such that it canbe substituted for an amino acid without substantially diminishing theability to react with T cell lines or clones. A nonpeptide mimetic is acompound that does not contain amino acids, and that has an overallconformation that is similar to a peptide, e.g. such that the ability ofthe mimetic to react with T cell lines or clones is not substantiallydiminished relative to the ability of a given peptide.

According to the invention, a variant, derivative, modified form,fragment, part or portion of an amino acid sequence, peptide or proteinpreferably has a functional property of the amino acid sequence, peptideor protein, respectively, from which it has been derived, i.e. it isfunctionally equivalent. In one embodiment, a variant, derivative,modified form, fragment, part or portion of an amino acid sequence,peptide or protein is immunologically equivalent to the amino acidsequence, peptide or protein, respectively, from which it has beenderived. In one embodiment, the functional property is an immunologicalproperty.

A particular property is the ability to form a complex with MHCmolecules and, where appropriate, generate an immune response,preferably by stimulating cytotoxic or T helper cells.

The term “immunologically equivalent” means that the immunologicallyequivalent molecule such as the immunologically equivalent amino acidsequence exhibits the same or essentially the same immunologicalproperties and/or exerts the same or essentially the same immunologicaleffects, e.g., with respect to the type of the immunological effect suchas induction of a humoral and/or cellular immune response, the strengthand/or duration of the induced immune reaction, or the specificity ofthe induced immune reaction. In the context of the present invention,the term “immunologically equivalent” is preferably used with respect tothe immunological effects or properties of a peptide or peptide variantused for immunization. For example, an amino acid sequence isimmunologically equivalent to a reference amino acid sequence if saidamino acid sequence when exposed to the immune system of a subjectinduces an immune reaction having a specificity of reacting with thereference amino acid sequence.

The term “derived” means according to the invention that a particularentity, in particular a particular sequence, is present in the objectfrom which it is derived, in particular an organism or molecule. In thecase of amino acid sequences, especially particular sequence regions,“derived” in particular means that the relevant amino acid sequence isderived from an amino acid sequence in which it is present.

The term “cell” or “host cell” preferably relates to an intact cell,i.e. a cell with an intact membrane that has not released its normalintracellular components such as enzymes, organelles, or geneticmaterial. An intact cell preferably is a viable cell, i.e. a living cellcapable of carrying out its normal metabolic functions. Preferably saidterm relates according to the invention to any cell which can betransfected with an exogenous nucleic acid. Preferably, the cell whentransfected with an exogenous nucleic acid and transferred to arecipient can express the nucleic acid in the recipient. The term “cell”includes bacterial cells; other useful cells are yeast cells, fungalcells or mammalian cells. Suitable bacterial cells include cells fromgram-negative bacterial strains such as strains of Escherichia coli,Proteus, and Pseudomonas, and gram-positive bacterial strains such asstrains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus.Suitable fungal cell include cells from species of Trichoderma,Neurospora, and Aspergillus. Suitable yeast cells include cells fromspecies of Saccharomyces (Tor example Saccharomyces cerevisiae),Schizosaccharomyces (for example Schizo saccharomyces pombe), Pichia(for example Pichia pastoris and Pichia methanolicd), and Hansenula.Suitable mammalian cells include for example CHO cells, BHK cells, HeLacells, COS cells, 293 HEK and the like. However, amphibian cells, insectcells, plant cells, and any other cells used in the art for theexpression of heterologous proteins can be used as well. Mammalian cellsare particularly preferred for adoptive transfer, such as cells fromhumans, mice, hamsters, pigs, goats, and primates. The cells may bederived from a large number of tissue types and include primary cellsand cell lines such as cells of the immune system, in particularantigen-presenting cells such as dendritic cells and T cells, stem cellssuch as hematopoietic stem cells and mesenchymal stem cells and othercell types. An antigen-presenting cell is a cell that displays antigenin the context of major histocompatibility complex on its surface. Tcells may recognize this complex using their T cell receptor (TCR).

A cell which comprises a nucleic acid molecule preferably express thepeptide or protein encoded by the nucleic acid.

The cell may be a recombinant cell and may secrete the encoded peptideor protein, may express it on the surface and preferably mayadditionally express an MHC molecule which binds to said peptide orprotein or a procession product thereof. In one embodiment, the cellexpresses the MHC molecule endogenously. In a further embodiment, thecell expresses the MHC molecule and/or the peptide or protein or theprocession product thereof in a recombinant manner. The cell ispreferably nonproliferative. In a preferred embodiment, the cell is anantigen-presenting cell, in particular a dendritic cell, a monocyte or amacrophage.

The term “clonal expansion” refers to a process wherein a specificentity is multiplied. In the context of the present invention, the termis preferably used in the context of an immunological response in whichlymphocytes are stimulated by an antigen, proliferate, and the specificlymphocyte recognizing said antigen is amplified. Preferably, clonalexpansion leads to differentiation of the lymphocytes.

A disease associated with antigen expression may be detected based onthe presence of T cells that specifically react with a peptide in abiological sample. Within certain methods, a biological samplecomprising CD4+ and/or CD8+ T cells isolated from a patient is incubatedwith a peptide of the invention, a nucleic acid encoding such peptideand/or an antigen-presenting cell that expresses and/or presents atleast an immunogenic portion of such a peptide, and the presence orabsence of specific activation of the T cells is detected. Suitablebiological samples include, but are not limited to, isolated T cells.For example, T cells may be isolated from a patient by routinetechniques (such as by Ficoll/Hypaque density gradient centrifugation ofperipheral blood lymphocytes). For CD4+ T cells, activation ispreferably detected by evaluating proliferation of the T cells. For CD8+T cells, activation is preferably detected by evaluating cytolyticactivity. A level of proliferation that is at least two fold greaterand/or a level of cytolytic activity that is at least 20% greater thanin disease-free subjects indicates the presence of a disease associatedwith antigen expression in the subject.

“Reduce” or “inhibit” as used herein means the ability to cause anoverall decrease, preferably of 5% or greater, 10% or greater, 20% orgreater, more preferably of 50% or greater, and most preferably of 75%or greater, in the level. The term “inhibit” or similar phrases includesa complete or essentially complete inhibition, i.e. a reduction to zeroor essentially to zero.

Terms such as “increase” or “enhance” preferably relate to an increaseor enhancement by about at least 10%, preferably at least 20%,preferably at least 30%, more preferably at least 40%, more preferablyat least 50%, even more preferably at least 80%, and most preferably atleast 100%.

The agents, compositions and methods described herein can be used totreat a subject with a disease, e.g., a disease characterized by thepresence of diseased cells expressing CLDN6 and preferably presentingCLDN6 in the context of MHC molecules. Examples of diseases which can betreated and/or prevented encompass all diseases expressing CLDN6.Particularly preferred diseases are cancer diseases.

The agents, compositions and methods described herein may also be usedfor immunization or vaccination to prevent a disease described herein.

The terms “normal tissue” or “normal conditions” refer to healthy tissueor the conditions in a healthy subject, i.e., non-pathologicalconditions, wherein “healthy” preferably means non-cancerous.

The term “disease” refers to an abnormal condition that affects the bodyof an individual. A disease is often construed as a medical conditionassociated with specific symptoms and signs. A disease may be caused byfactors originally from an external source, such as infectious disease,or it may be caused by internal dysfunctions, such as autoimmunediseases. In humans, “disease” is often used more broadly to refer toany condition that causes pain, dysfunction, distress, social problems,or death to the individual afflicted, or similar problems for those incontact with the individual. In this broader sense, it sometimesincludes injuries, disabilities, disorders, syndromes, infections,isolated symptoms, deviant behaviors, and atypical variations ofstructure and function, while in other contexts and for other purposesthese may be considered distinguishable categories. Diseases usuallyaffect individuals not only physically, but also emotionally, ascontracting and living with many diseases can alter one's perspective onlife, and one's personality. According to the invention, the term“disease” includes cancer, in particular those forms of cancer describedherein. Any reference herein to cancer or particular forms of canceralso includes cancer metastasis thereof. In a preferred embodiment, adisease to be treated according to the present application involvescells expressing CLDN6 and optionally presenting CLDN6 in the context ofMHC molecules.

“Diseases involving cells expressing CLDN6” or similar expressions meansaccording to the invention that CLDN6 is expressed in cells of adiseased tissue or organ. In one embodiment, expression of CLDN6 incells of a diseased tissue or organ is increased compared to the statein a healthy tissue or organ. An increase refers to an increase by atleast 10%, in particular at least 20%, at least 50%, at least 100%, atleast 200%, at least 500%, at least 1000%, at least 10000% or even more.In one embodiment, expression is only found in a diseased tissue, whileexpression in a healthy tissue is repressed. According to the invention,diseases involving cells expressing CLDN6 include cancer diseases.Furthermore, according to the invention, cancer diseases preferably arethose wherein the cancer cells express CLDN6.

The terms “cancer disease” or “cancer” refer to or describe thephysiological condition in an individual that is typically characterizedby unregulated cell growth. Examples of cancers include, but are notlimited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticularly, examples of such cancers include bone cancer, bloodcancer, lung cancer, liver cancer, pancreatic cancer, skin cancer,cancer of the head or neck, cutaneous or intraocular melanoma, uterinecancer, ovarian cancer, rectal cancer, cancer of the anal region,stomach cancer, colon cancer, breast cancer, prostate cancer, uterinecancer, carcinoma of the sexual and reproductive organs, Hodgkin'sDisease, cancer of the esophagus, cancer of the small intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the bladder, cancer of the kidney, renal cell carcinoma,carcinoma of the renal pelvis, neoplasms of the central nervous system(CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma,and pituitary adenoma. The term “cancer” according to the invention alsocomprises cancer metastases. Preferably, a “cancer disease” ischaracterized by cells expressing CLDN6 and a cancer cell expressesCLDN6.

A diseased cell preferably is a cell expressing CLDN6 said CLDN6preferably being present on the surface of said cell as transmembraneprotein and/or being presented by said cell in the context of MHC suchas MHC I. A cell expressing CLDN6 preferably is a cancer cell,preferably of the cancers described herein.

In one embodiment, a cancer disease is a malignant disease which ischaracterized by the properties of anaplasia, invasiveness, andmetastasis. A malignant tumor may be contrasted with a non-cancerousbenign tumor in that a malignancy is not self-limited in its growth, iscapable of invading into adjacent tissues, and may be capable ofspreading to distant tissues (metastasizing), while a benign tumor hasnone of those properties.

According to the invention, the term “tumor” or “tumor disease” refersto a swelling or lesion formed by an abnormal growth of cells (calledneoplastic cells or tumor cells). By “tumor cell” is meant an abnormalcell that grows by a rapid, uncontrolled cellular proliferation andcontinues to grow after the stimuli that initiated the new growth cease.Tumors show partial or complete lack of structural organization andfunctional coordination with the normal tissue, and usually form adistinct mass of tissue, which may be either benign, pre-malignant ormalignant.

According to the invention, a “carcinoma” is a malignant tumor derivedfrom epithelial cells. This group represents the most common cancers,including the common forms of breast, prostate, lung and colon cancer.

“Adenocarcinoma” is a cancer that originates in glandular tissue. Thistissue is also part of a larger tissue category known as epithelialtissue. Epithelial tissue includes skin, glands and a variety of othertissue that lines the cavities and organs of the body. Epithelium isderived embryologically from ectoderm, endoderm and mesoderm. To beclassified as adenocarcinoma, the cells do not necessarily need to bepart of a gland, as long as they have secretory properties. This form ofcarcinoma can occur in some higher mammals, including humans. Welldifferentiated adenocarcinomas tend to resemble the glandular tissuethat they are derived from, while poorly differentiated may not. Bystaining the cells from a biopsy, a pathologist will determine whetherthe tumor is an adenocarcinoma or some other type of cancer.Adenocarcinomas can arise in many tissues of the body due to theubiquitous nature of glands within the body. While each gland may not besecreting the same substance, as long as there is an exocrine functionto the cell, it is considered glandular and its malignant form istherefore named adenocarcinoma. Malignant adenocarcinomas invade othertissues and often metastasize given enough time to do so. Ovarianadenocarcinoma is the most common type of ovarian carcinoma. It includesthe serous and mucinous adenocarcinomas, the clear cell adenocarcinomaand the endometrioid adenocarcinoma.

Lymphoma and leukemia are malignancies derived from hematopoietic(blood-forming) cells.

Blastic tumor or blastoma is a tumor (usually malignant) which resemblesan immature or embryonic tissue. Many of these tumors are most common inchildren.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor at the target site depends onangiogenesis. Tumor metastasis often occurs even after the removal ofthe primary tumor because tumor cells or components may remain anddevelop metastatic potential. In one embodiment, the term “metastasis”according to the invention relates to “distant metastasis” which relatesto a metastasis which is remote from the primary tumor and the regionallymph node system. In one embodiment, the term “metastasis” according tothe invention relates to lymph node metastasis.

The cells of a secondary or metastatic tumor are like those in theoriginal tumor. This means, for example, that, if ovarian cancermetastasizes to the liver, the secondary tumor is made up of abnormalovarian cells, not of abnormal liver cells. The tumor in the liver isthen called metastatic ovarian cancer, not liver cancer.

A relapse or recurrence occurs when a person is affected again by acondition that affected them in the past. For example, if a patient hassuffered from a tumor disease, has received a successful treatment ofsaid disease and again develops said disease said newly developeddisease may be considered as relapse or recurrence. However, accordingto the invention, a relapse or recurrence of a tumor disease may butdoes not necessarily occur at the site of the original tumor disease.Thus, for example, if a patient has suffered from ovarian tumor and hasreceived a successful treatment a relapse or recurrence may be theoccurrence of an ovarian tumor or the occurrence of a tumor at a sitedifferent to ovary. A relapse or recurrence of a tumor also includessituations wherein a tumor occurs at a site different to the site of theoriginal tumor as well as at the site of the original tumor. Preferably,the original tumor for which the patient has received a treatment is aprimary tumor and the tumor at a site different to the site of theoriginal tumor is a secondary or metastatic tumor.

The term “treatment” or “therapeutic treatment” relates to any treatmentwhich improves the health status and/or prolongs (increases) thelifespan of an individual. Said treatment may eliminate the disease inan individual, arrest or slow the development of a disease in anindividual, inhibit or slow the development of a disease in anindividual, decrease the frequency or severity of symptoms in anindividual, and/or decrease the recurrence in an individual whocurrently has or who previously has had a disease.

The terms “prophylactic treatment” or “preventive treatment” relate toany treatment that is intended to prevent a disease from occurring in anindividual. The terms “prophylactic treatment” or “preventive treatment”are used herein interchangeably.

The terms “individual” and “subject” are used herein interchangeably.They refer to human beings, non-human primates or other mammals (e.g.mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate)that can be afflicted with or are susceptible to a disease or disorder(e.g., cancer) but may or may not have the disease or disorder. In manyembodiments, the individual is a human being. Unless otherwise stated,the terms “individual” and “subject” do not denote a particular age, andthus encompass adults, elderlies, children, and newborns. In preferredembodiments of the present invention, the “individual” or “subject” is a“patient”. The term “patient” means according to the invention a subjectfor treatment, in particular a diseased subject.

By “being at risk” is meant a subject, i.e. a patient, that isidentified as having a higher than normal chance of developing adisease, in particular cancer, compared to the general population. Inaddition, a subject who has had, or who currently has, a disease, inparticular cancer is a subject who has an increased risk for developinga disease, as such a subject may continue to develop a disease. Subjectswho currently have, or who have had, a cancer also have an increasedrisk for cancer metastases.

The term “immunotherapy” relates to a treatment involving a specificimmune reaction.

In the context of the present invention, terms such as “protect”,“prevent”, “prophylactic”, “preventive”, or “protective” relate to theprevention or treatment or both of the occurrence and/or the propagationof a disease in a subject and, in particular, to minimizing the chancethat a subject will develop a disease or to delaying the development ofa disease. For example, a person at risk for a tumor, as describedabove, would be a candidate for therapy to prevent a tumor.

A prophylactic administration of an immunotherapy, for example, aprophylactic administration of an agent or composition of the invention,preferably protects the recipient from the development of a disease. Atherapeutic administration of an immunotherapy, for example, atherapeutic administration of an agent or composition of the invention,may lead to the inhibition of the progress/growth of the disease. Thiscomprises the deceleration of the progress/growth of the disease, inparticular a disruption of the progression of the disease, whichpreferably leads to elimination of the disease.

Immunotherapy may be performed using any of a variety of techniques, inwhich agents provided herein preferably function to removeCLDN6-expressing cells from a patient. Such removal may take place as aresult of enhancing or inducing an immune response in a patient specificfor CLDN6 or a cell expressing CLDN6 and/or presenting CLDN6 in thecontext of MHC molecules.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against diseased cells with theadministration of immune response-modifying agents (such as peptides andnucleic acids as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells) that can directly orindirectly mediate antitumor effects and does not necessarily depend onan intact host immune system. Examples of effector cells include Tlymphocytes (such as CD8+ cytotoxic T lymphocytes and CD4+T-helperlymphocytes), and antigen-presenting cells (such as dendritic cells andmacrophages). T cell receptors specific for the CLDN6 peptides recitedherein and artificial T cell receptors specific for CLDN6 may betransferred into effector cells for adoptive immunotherapy.

As noted above, immunoreactive peptides as provided herein may be usedto rapidly expand antigen-specific T cell cultures in order to generatea sufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic cells, macrophages,monocytes, fibroblasts and/or B cells, may be pulsed with immunoreactivepeptides or transfected with one or more nucleic acids using standardtechniques well known in the art. Cultured effector cells for use intherapy must be able to grow and distribute widely, and to survive longterm in vivo. Studies have shown that cultured effector cells can beinduced to grow in vivo and to survive long term in substantial numbersby repeated stimulation with antigen supplemented with IL-2 (see, forexample, Cheever et al. (1997), Immunological Reviews 157, 177.

Alternatively, a nucleic acid expressing a peptide recited herein may beintroduced into antigen-presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.

Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration.

Methods disclosed herein may involve the administration of autologous Tcells that have been activated in response to a peptide orpeptide-expressing antigen presenting cell. Such T cells may be CD4+and/or CD8+, and may be proliferated as described above. The T cells maybe administered to the subject in an amount effective to inhibit thedevelopment of a disease.

The term “immunization” or “vaccination” describes the process oftreating a subject with the purpose of inducing an immune response fortherapeutic or prophylactic reasons.

The term “in vivo” relates to the situation in a subject.

According to the invention, a “sample” may be any sample usefulaccording to the present invention, in particular a biological samplesuch a tissue sample, including body fluids, and/or a cellular sampleand may be obtained in the conventional manner such as by tissue biopsy,including punch biopsy, and by taking blood, bronchial aspirate, sputum,urine, feces or other body fluids. According to the invention, the term“sample” also includes processed samples such as fractions or isolatesof biological samples, e.g. nucleic acid and peptide/protein isolates.

The compounds and agents described herein may be administered in theform of any suitable pharmaceutical composition.

The pharmaceutical compositions of the invention are preferably sterileand contain an effective amount of the agents described herein andoptionally of further agents as discussed herein to generate the desiredreaction or the desired effect.

Pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. A pharmaceuticalcomposition may e.g. be in the form of a solution or suspension.

A pharmaceutical composition may comprise salts, buffer substances,preservatives, carriers, diluents and/or excipients all of which arepreferably pharmaceutically acceptable. The term “pharmaceuticallyacceptable” refers to the non-toxicity of a material which does notinteract with the action of the active component of the pharmaceuticalcomposition.

Salts which are not pharmaceutically acceptable may be used forpreparing pharmaceutically acceptable salts and are included in theinvention. Pharmaceutically acceptable salts of this kind comprise in anon limiting way those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallyacceptable salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

Suitable buffer substances for use in a pharmaceutical compositioninclude acetic acid in a salt, citric acid in a salt, boric acid in asalt and phosphoric acid in a salt.

Suitable preservatives for use in a pharmaceutical composition includebenzalkonium chloride, chlorobutanol, paraben and thimerosal.

An injectible formulation may comprise a pharmaceutically acceptableexcipient such as Ringer Lactate.

The term “carrier” refers to an organic or inorganic component, of anatural or synthetic nature, in which the active component is combinedin order to facilitate, enhance or enable application. According to theinvention, the term “carrier” also includes one or more compatible solidor liquid fillers, diluents or encapsulating substances, which aresuitable for administration to a patient.

Possible carrier substances for parenteral administration are e.g.sterile water, Ringer, Ringer lactate, sterile sodium chloride solution,polyalkylene glycols, hydrogenated naphthalenes and, in particular,biocompatible lactide polymers, lactide/glycolide copolymers orpolyoxyethylene/polyoxy-propylene copolymers.

The term “excipient” when used herein is intended to indicate allsubstances which may be present in a pharmaceutical composition andwhich are not active ingredients such as, e.g., carriers, binders,lubricants, thickeners, surface active agents, preservatives,emulsifiers, buffers, flavoring agents, or colorants.

The agents and compositions described herein may be administered via anyconventional route, such as by parenteral administration including byinjection or infusion. Administration is preferably parenterally, e.g.intravenously, intraarterially, subcutaneously, intradermally orintramuscularly.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The agents and compositions described herein are administered ineffective amounts. An “effective amount” refers to the amount whichachieves a desired reaction or a desired effect alone or together withfurther doses. In the case of treatment of a particular disease or of aparticular condition, the desired reaction preferably relates toinhibition of the course of the disease. This comprises slowing down theprogress of the disease and, in particular, interrupting or reversingthe progress of the disease. The desired reaction in a treatment of adisease or of a condition may also be delay of the onset or a preventionof the onset of said disease or said condition.

An effective amount of an agent or composition described herein willdepend on the condition to be treated, the severeness of the disease,the individual parameters of the patient, including age, physiologicalcondition, size and weight, the duration of treatment, the type of anaccompanying therapy (if present), the specific route of administrationand similar factors. Accordingly, the doses administered of the agentsdescribed herein may depend on various of such parameters. In the casethat a reaction in a patient is insufficient with an initial dose,higher doses (or effectively higher doses achieved by a different, morelocalized route of administration) may be used.

The agents and compositions described herein can be administered topatients, e.g., in vivo, to treat or prevent a variety of disorders suchas those described herein. Preferred patients include human patientshaving disorders that can be corrected or ameliorated by administeringthe agents and compositions described herein. This includes disordersinvolving cells characterized by expression of CLDN6.

For example, in one embodiment, agents described herein can be used totreat a patient with a cancer disease, e.g., a cancer disease such asdescribed herein characterized by the presence of cancer cellsexpressing CLDN6.

The pharmaceutical compositions and methods of treatment describedaccording to the invention may also be used for immunization orvaccination to prevent a disease described herein.

The pharmaceutical composition of the invention may be administeredtogether with supplementing immunity-enhancing substances such as one ormore adjuvants and may comprise one or more immunity-enhancingsubstances to further increase its effectiveness, preferably to achievea synergistic effect of immunostimulation. The term “adjuvant” relatesto compounds which prolongs or enhances or accelerates an immuneresponse. Various mechanisms are possible in this respect, depending onthe various types of adjuvants. For example, compounds which allow thematuration of the DC, e.g. lipopolysaccharides or CD40 ligand, form afirst class of suitable adjuvants. Generally, any agent which influencesthe immune system of the type of a “danger signal” (LPS, GP96, dsRNAetc.) or cytokines, such as GM-CSF, can be used as an adjuvant whichenables an immune response to be intensified and/or influenced in acontrolled manner CpG oligodeoxynucleotides can optionally also be usedin this context, although their side effects which occur under certaincircumstances, as explained above, are to be considered. Particularlypreferred adjuvants are cytokines, such as monokines, lymphokines,interleukins or chemokines, e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-12, IFNα, IFNγ, GM-CSF, LT-α, or growthfactors, e.g. hGH. Further known adjuvants are aluminium hydroxide,Freund's adjuvant or oil such as Montanide®, most preferred Montanide®ISA51. Lipopeptides, such as Pam3Cys, are also suitable for use asadjuvants in the pharmaceutical composition of the present invention.

The pharmaceutical composition can be administered locally orsystemically, preferably systemically.

The term “systemic administration” refers to the administration of anagent such that the agent becomes widely distributed in the body of anindividual in significant amounts and develops a desired effect. Forexample, the agent may develop its desired effect in the blood and/orreaches its desired site of action via the vascular system. Typicalsystemic routes of administration include administration by introducingthe agent directly into the vascular system or oral, pulmonary, orintramuscular administration wherein the agent is adsorbed, enters thevascular system, and is carried to one or more desired site(s) of actionvia the blood.

According to the present invention, it is preferred that the systemicadministration is by parenteral administration. The term “parenteraladministration” refers to administration of an agent such that the agentdoes not pass the intestine. The term “parenteral administration”includes intravenous administration, subcutaneous administration,intradermal administration or intraarterial administration but is notlimited thereto.

Administration may also be carried out, for example, orally,intraperitonealy or intramuscularly.

The agents and compositions provided herein may be used alone or incombination with conventional therapeutic regimens such as surgery,irradiation, chemotherapy and/or bone marrow transplantation(autologous, syngeneic, allogeneic or unrelated).

The present invention is described in detail by the figures and examplesbelow, which are used only for illustration purposes and are not meantto be limiting. Owing to the description and the examples, furtherembodiments which are likewise included in the invention are accessibleto the skilled worker.

FIGURES

FIG. 1: Representation of the TCR-CD3 complex. The intracytoplasmic CD3immunoreceptor tyrosine-based activation motifs (ITAMs) are indicated ascylinders (adapted from “The T cell receptor facts book”, MP Lefranc, GLefranc, 2001).

FIG. 2: The design of successive generations of CARs. Schematicrepresentation of the different generations of CARs (1G, firstgeneration, 2G, second generation, 3G, third generation). The firstgeneration contains extracellular scFvs and the cytoplasmic CD3 ζchain/ZAP70 mediating cytotoxicity, the second generation additionallyCD28/PI3K promoting proliferation and the third generation furthermore4-1BB or OX40/TRAF sustaining cell survival (Casucci, M. et al. (2011)2: 378-382).

FIG. 3: Schematic representation of the different receptor formats forthe redirection of T cells against CLDN6. Left: a second generation CARconsisting of a CLDN6-specific scFv fragment, a IgG1-derived spacerdomain, a CD28 costimulatory and a CD3ζ signaling domain (CAR-28ζ);middle: a novel CAR format based on the linkage of the scFv with theconstant domain of the murine TCRβ chain and coexpression of theconstant domain of the murine TCRα chain (CAR/Cα); right: a murine TCRcomposed of TCR α/β chains (mu, murine TCR);

FIG. 4: Claudin-6 expression in normal tissues and different cancers.The CLDN6 mRNA expression was analyzed by qRT-PCR in different normaltissue and 47 ovarian carcinoma specimens.

FIG. 5. Technology platform for TCR isolation and validation. Theapproach integrates all steps from isolation of antigen-specific T cells(top) to TCR cloning (middle) and TCR validation (bottom).HLA-A2/DR1-transgenic mice are immunized with tumor antigen encodingmRNA. Spleen cells of these mice are analyzed for ex vivo reactivityagainst the respective antigen by IFNγ-ELISPOT and antigen-specificmurine CD8+ T cells are isolated after in vitro restimulation based onactivation-induced expression of CD137 by flow cytometry (top). Singlecells are harvested in multiwell-plates and subjected to first-strandcDNA synthesis and enrichment by a global PCR amplification step. TCRα/β variable regions are cloned into vectors for in vitro transcription(IVT) containing the constant region cassettes (middle). TCR α/β chainRNAs are transferred into human CD8+ T cells, cocultured with APCsexpressing the appropriate antigen and HLA molecules and tested forfunctional reprogramming of engineered T cells (bottom).

FIG. 6: Ex vivo reactivity of spleen cells from immunizedHLA-A*02-transgenic mice against CLDN6-derived peptides analyzed byIFNγ-ELISPOT assay. HLA-A*02 CLDN6-specific binding peptides werepredicted applying a specific algorithm (Rammensee H. et al. (1999)Immunogenetics 50, 213-9). Spleen cells were analyzed for reactivityagainst CLDN6 peptide pool or predicted HLA-A*02-binding CLDN6-derivedpeptides A2-1-6. Positive control: PMA-treated spleen cells; negativecontrol: an irrelevant peptide pool (HIV-gag), irrelevant nonamerpeptide (PLAC1-31-39).

FIG. 7: Flow cytometry sorting of CLDN6-specific murine CD8+ T cellsfrom HLA-A*02-transgenic mice after in-vitro restimulation. SingleCD8+/CD137+ T cells were isolated by flow cytometry and harvested inmultiwell plates for TCR cloning after restimulation of spleen cellswith CLDN6 overlapping peptide pool. Control: spleen cells restimulatedwith irrelevant peptide pool.

FIG. 8: Specificity testing of TCRs isolated from CD8+ T cells ofCLDN6-immunized mice. CD8+ T cells of a HLA-A*02-positive healthy donorwere transfected with TCR-α/β chain RNAs and tested for recognition ofK562-A2 cells transfected with CLDN6 RNA or pulsed with CLDN6overlapping 15mer peptides (=Cl6 pool) or CLDN6 HLA-A*02 bindingpeptides (Cl6-A2-1, Cl6-A2-2) by IFNγ-ELISPOT. Negative controls:irrelevant peptide pool, irrelevant 9mer peptide; Positive control: SEB

FIG. 9: Surface expression of CLDN6-specific murine TCRs on humanpreactivated CD8+ T cells. CD8+ T cells were preactivated with OKT3 andtransfected with 20 μg TCR α/β RNA. 20 h after electroporation cellswere stained with a PE-conjugated anti-CD8 antibody and APC-conjugatedantibody recognizing the murine constant domain of the TCR 13 chain.Cells were gated on single lymphocytes.

FIG. 10: Tumor cell lysis mediated by CLDN6-specific TCRs. PreactivatedCD8+ T cells were transfected with 20 μg TCR α/β RNAs and cocultured 20h later together with HLA-A*02-expressing CLDN6-positive (PA1-Luc;NIH-OvCar3) or -negative (SK-Mel-37) tumor cell lines with an E:T(effector cell: target cell) ratio of 30:1. Specific lysis was analyzedby luciferase-based cytotoxicity assay after 4 h coculture.

FIG. 11: Dose-dependent proliferation mediated by CLDN6-specific TCRs inresponse to CLDN6-expressing target cells. CD8+ T cells were transfectedwith 20 μg TCR RNA, labeled with CFSE and cocultured with autologousmonocytes transfected with titrated amounts of CLDN6 RNA. After 4 daysof coculture cells were stained with an APC-Cy7-labeled anti-CD8antibody. A) Specific proliferation was analyzed by flowcytometry basedon the dilution of the CFSE proliferation dye. Dotplots show livingCD8+T lymphocytes after coculture with monocytes transfected with 1 μgCLDN6-RNA. B) Bars show the percentage of proliferating CD8+ T cells.

FIG. 12: Surface expression of CLDN6-specific CAR constructs on restinghuman CD4+ and CD8+ T cells. PBMCs were transfected with 10 μg CAR RNA.20 h after electroporation cells were stained with a PE-conjugatedanti-CD8, a FITC-conjugated anti-CD4 and an idiotype-specific antibodylabeled with Dylight-650. Cells were gated on single CD4+ or CD8+ Tcells.

FIG. 13: Tumor cell lysis mediated by different CLDN-6 targetingreceptor formats. Preactivated CD8+ T cells were transfected with CAR orTCR RNAs and cocultured 20 h later together with CLDN6-positive orCLDN6-negative tumor cell lines PA1 and MDA-MB-231-Luc at different E:Tratios. Specific lysis was analyzed by luciferase-based cytotoxicityassay after 4 h coculture.

FIG. 14: Antigen-specific proliferation mediated by CLDN6-specific CARin response to CLDN6-expressing target cells. CD8+ T cells weretransfected with 20 μg TCR or CAR RNA, labeled with CFSE and coculturedwith autologous iDC transfected with CLDN6 or control RNA for 4 days. A)TCR/CAR surface expression was analyzed by flow cytometry after stainingwith a murine APC-conjugated TCRβ-specific or a Dylight650-conjugatedidiotype-specific antibody. Specific proliferation was analyzed by flowcytometry based on the dilution of the CFSE proliferation dye.

FIG. 15: Surface expression of different mutants of CLDN6-CAR-28ζconstructs with mutated cysteine 46 on preactivated CD8+ T cells. CD8+ Tcells were preactivated with OKT3 and transfected with 20 μg CAR RNA. 20h after electroporation cells were stained with a PE-conjugated anti-CD8antibody and an idiotype-specific antibody labeled with Dylight650.Cells were gated on singlets and lymphocytes.

FIG. 16: Surface expression of different mutants of CAR-28ζ constructswith mutated cysteine 46 on preactivated CD8+ T cells of three differentdonors. CD8+ T cells were preactivated with OKT3 and transfected with 20μg CAR RNA. 20 h after electroporation cells were stained with anidiotype-specific antibody labeled with Dylight650. Cells were gated onCAR-expressing CD8+T lymphocytes. The results of three independentexperiments are shown. Top: the percentage of CAR+/CD8+ T cells isshown; bottom: the mean fluorescence intensity of CAR-positive CD8+ Tcells is shown;

FIG. 17: Specific tumor cell lysis mediated by different mutants ofCLDN6-CAR-28ζ constructs with mutated cysteine 46. A) The CLDN6 surfaceexpression on target cell lines was analyzed after staining with aAlexa647-conjugated CLDN6-specific antibody by flow cytometry. B)Preactivated CD8+ T cells were transfected with 20 μg CAR RNA andcocultured 20 h later together with CLDN6-positive (PA1) orCLDN6-negative (MDA-MB-231-Luc-Tomato) tumor cell lines at different E:Tratios. Specific lysis was analyzed by luciferase-based cytotoxicityassay after 4 h coculture. C) CAR surface expression on T cells wasanalyzed after staining with a fluorochrome-conjugated CD8-specific andan idiotype-specific antibody by flow cytometry.

FIG. 18: Dose-dependent lysis of target cells mediated by differentmutants of CLDN6-CAR-28ζ constructs with mutated cysteine 46. A)Preactivated CD8+ T cells were transfected with 20 μg CAR RNA andcocultured 20 h later together with autologous iDC transfected withtitrated amounts of CLDN6-RNA (E:T=30:1). B) The CLDN6 surfaceexpression on transfected iDCs was analyzed after staining with aAlexa647-conjugated CLDN6-specific antibody by flowcytometry.

FIG. 19: Schematic representation of the retroviral SIN construct usedfor stable CAR expression. The plasmid pES12.6-CLDN6-CAR-C46S was usedfor transient generation of GALV-enveloped SIN-vector using HEK293Tcells.

FIG. 20: Detection of CLDN6-CAR and CAR against an unrelated tumorantigen on transduced human T cells used for adoptive transfer into NSGmice. Cells were stained with fluorochrome-conjugated antibodies (BDBiosciences) directed against CD8 and CD4 as well as withidiotype-specific antibodies directed against the respective scFv partof the CLDN6-CAR (anti-IMAB206, Ganymed Pharmaceuticals AG) and the CARagainst an unrelated tumor antigen, respectively. Cells were gated onsingle CD8⁺ or CD4⁺ lymphocytes. Transduced T cells were used foradoptive cell transfer in OV90-SC12-engrafted NSG mice. The transductionrate for the CLDN6-CAR and the CAR against an unrelated tumor antigenwas about 37% of CD4⁺ and 20% of CD8⁺ as well as 36% of CD4⁺ and 24 ofCD8⁺ cells, respectively. Graphs are displayed in logarithmic scale.

FIG. 21: Anti-tumoral activity of CLDN6-CAR transduced T cells in anovarian carcinoma model. 1×10⁷ human OV90-SC12 tumor cells (ATCCCRL11732) were injected subcutaneously into NSG mice (10 mice/group).After 4 days, the mice were treated with a single intravenous injectionof 1×10⁷ CD3/CD28 bead stimulated, retrovirally transduced human T cells(about 37% of CD4 and 20% of CD8 were CLDN6-CAR positive). A) Scheme ofthe experimental set up. B) Delay of tumor growth in CLDN6-CAR treatedmice compared to control groups (no T cells, untransduced T cells, and Tcells transduced with CAR against an unrelated tumor antigen). Tumormonitoring by volume measurements and analysis of peripheral blood wasperformed weekly. Results are expressed as mean tumor volume±SEM withn=10 mice for all groups. Tumor volume was calculated using thefollowing formula: V=½*(length*square width). The plot for the CLDN6-CARtreated mice is significantly different from the control treatment groupfor t=31 days (*ANOVA, P<0.05). C) Tumor-growth curves of the individualmice of each group are shown. Please note, 2 mice in the unrelated tumorantigen group had to be sacrificed on day 24 due to high tumor burden(marked with +).

FIG. 22. Proliferation of CAR T cells after co-culture with CLND6expressing iDCs. CD8⁺ T cells were transfected with IVT-RNA encoding aCAR directed against A) CLDN6 or B) an unrelated tumor antigen asnegative control, labeled with CFSE (carboxyfluorescein succinimidylester) and cocultured with CLDN6-transfected autologous iDCs for 4 days.Proliferation of CAR T cells was analyzed based on the dilution of CFSEby flowcytometry. Cells were gated on single living CD8⁺ T lymphocytes.

EXAMPLES

The techniques and methods used herein are described herein or carriedout in a manner known per se and as described, for example, in Sambrooket al., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Allmethods including the use of kits and reagents are carried out accordingto the manufacturers' information unless specifically indicated.

Example 1: Materials and Methods

Cell Lines and Reagents

The human chronic myeloid leukemia cell line K562 (Lozzio, C. B. &Lozzio, B. B (1975), Blood 45, 321-334) was cultured under standardconditions. K562 cells stably transfected with HLA-A*0201 (Britten, C.M. et al. (2002), J. Immunol. Methods 259, 95-110) (referred to e.g. asK562-A*0201) were used for validation assays. The primary human newbornforeskin fibroblast cell line CCD-1079Sk (ATCC No. CRL-2097) wascultured according to the manufacturers' instructions.

The human CLDN6 expressing ovarian carcinoma cell line OV-90-SC12 wasused for in vivo validation of the CLDN6-CAR.

The culture medium for PA-1-SC12_A0201 luc_gfp_F7 is composed of 86%RPMI 1640+ Glutamax (Co. Gibco, Cat-No. 61870), 10% FCS (Co. Biochrome,Cat-No. S0615), 1% Sodium Pyruvate (100 mM) (Co. Gibco, Cat-No. 11360),1% MEM Non-Essential Amino Acids Solution (100×) (Co. Gibco, Cat-No.11140), 2% Sodium Bicarbonate 7.5% solution (Co. Gibco, Cat-No. 25080).

The culture medium for OV-90-SC12 is composed of 41.5% MCDB 105 (Co.Sigma Aldrich, Cat-No. M6395-1L), 41.5% Medium 199 (Co. Sigma Aldrich,Cat-No. M2154-500 mL), 15% FCS (Co. Biochrome, Cat-No. S0615), 2% SodiumBicarbonate 7.5% solution (Co. Gibco, Cat-No. 25080).

The culture medium for SK-MEL-37 is composed of 90% DMEM+ Glutamax (Co.Gibco, Cat-No. 31966), 10% FCS (Co. Biochrome, Cat-No. 50615). Theculture medium for MDA-MB-231_luc_tom is composed of 88% RPMI 1640+Glutamax (Co. Gibco, Cat-No. 61870), 10% FCS (Co. Biochrome, Cat-No.S0615), 1% Sodium Pyruvate (100 mM) (Co. Gibco, Cat-No. 11360), 1% MEMNon-Essential Amino Acids Solution (100×) (Co. Gibco, Cat-No. 11140).Feeding and/or splitting of the cell lines was done every 2 to 3 days.

Peripheral Blood Mononuclear Cells (PBMCs), Monocytes and DendriticCells (DCs)

PBMCs were isolated by Ficoll-Hypaque (Amersham Biosciences, Uppsala,Sweden) density gradient centrifugation from buffy coats. HLAallelotypes were determined by PCR standard methods. Monocytes wereenriched with anti-CD14 microbeads (Miltenyi Biotech, Bergisch-Gladbach,Germany). Immature DCs (iDCs) were obtained by differentiating monocytesfor 5 days in cytokine-supplemented culture medium as described inKreiter et al. (2007), Cancer Immunol. Immunother., CII, 56, 1577-87.

Peptides and Peptide Pulsing of Stimulator Cells

Pools of N- and C-terminally free 15-mer peptides with 11 amino acidoverlaps corresponding to sequences of Claudin-6 or HIV-gag (referred toas antigen peptide pool) were synthesized by standard solid phasechemistry (JPT GmbH, Berlin, Germany) and dissolved in DMSO to a finalconcentration of 0.5 mg/ml. Nonamer peptides were reconstituted in PBS10% DMSO. For pulsing stimulator cells were incubated for 1 h at 37° C.in culture medium using different peptide concentrations.

Vectors for In Vitro Transcription (IVT) of RNA

All constructs are variants of the previously describedpST1-sec-insert-2βgUTR-A(120)-Sap1 plasmid (Holtkamp, S. et al. (2006),Blood 108, 4009-4017). To obtain plasmids encoding human TCR chains,cDNA coding for TCR-α or TCR-β₁ and TCR-β₂ constant regions wereamplified from human CD8+ T cells and cloned into this backbone. Forgeneration of plasmids encoding murine TCR chains, cDNAs coding forTCR-α, -β₁ and -β₂ constant regions were ordered from a commercialprovider and cloned analogously (GenBank accession numbers M14506,M64239 and X67127, respectively). Specific V(D)J PCR products wereintroduced into such cassettes to yield full-length TCR chains (referredto as pST1-human/murineTCRαβ-2βgUTR-A(120)).

Analogously, individual HLA class I and II alleles cloned from PBMCs ofdonors and beta-2-microglobulin (B2M) cDNA from human DCs were insertedinto this backbone (referred to as pST1-HLA class I/II-2βgUTR-A(120) andpST1-B2M-2βgUTR-A(120)).

Plasmids coding for pp65 antigen of CMV(pST1-sec-pp65-MITD-2βgUTR-A(120)) and NY-ESO-I(pST1-sec-NY-ESO-1-MITD-2βgUTR-A(120)) linked to a secretion signal(sec) and the MHC class I trafficking signal (MITD) were describedpreviously (Kreiter, S. et al. (2008), J. Immunol. 180, 309-318). PLAC1encoding plasmid pST1-sec-PLAC1-MITD-2βgUTR-A(120) was generated bycloning a cDNA obtained from a commercial provider (GenBank accessionnumber NM_021796) into the Kreiter et al. backbone. TPTE encodingplasmids pST1-αgUTR-TPTE-2βgUTR-A(120) andpST1-αgUTR-TPTE-MITD-2βgUTR-A(120) were generated by cloning a cDNAobtained from a commercial provider (GenBank accession number AF007118)into a variant of the Holtkamp et al. vector featuring an additionalalpha-globin 5′-untranslated region.

Primers were purchased from Operon Biotechnologies, Cologne, Germany.

Generation of In Vitro Transcribed (IVT) RNA and Transfer into Cells

Generation of IVT RNA was performed as described previously (Holtkamp,S. et al. (2006), Blood 108, 4009-4017) and added to cells suspended inX-VIVO 15 medium (Lonza, Basel, Switzerland) in a pre-cooled 4-mm gapsterile electroporation cuvette (Bio-Rad Laboratories GmbH, Munich,Germany) Electroporation was performed with a Gene-Pulser-II apparatus(Bio-Rad Laboratories GmbH, Munich, Germany) (T cells: 450 V/250 μF;IVSB T cells: 350 V/200 μF; SupT1 (ATCC No. CRL-1942): 300 V/200 μF;human DC: 300 V/150 μF; K562: 200 V/300 μF).

In Vivo Priming of T Cells by Intranodal Immunization of HLA A2.1/DR1Mice with IVT RNA

T cells of A2/DR1 mice (Pajot A. et al. (2004), Eur. J. Immunol. 34,3060-69) were primed in vivo against the antigen of interest byrepetitive intranodal immunization using antigen-encoding IVT RNA(Kreiter S. et al. (2010), Cancer Research 70, 9031-40). For intranodalimmunizations, mice were anesthetized with xylazine/ketamine Theinguinal lymph node was surgically exposed, 10 μL RNA (20 μg) diluted inRinger's solution and Rnase-free water were injected slowly using asingle-use 0.3-ml syringe with an ultrafine needle (31G, BDBiosciences), and the wound was closed. After six immunization cyclesthe mice were sacrificed and spleen cells were isolated.

Harvest of Spleen Cells

Following their dissection under sterile conditions, the spleens weretransferred to PBS containing falcon tubes. The spleens weremechanically disrupted with forceps and the cell suspensions wereobtained with a cell strainer (40 μm). The splenocytes were washed withPBS centrifuged and resuspended in a hypotonic buffer for lysis of theerythrocytes. After 5 min incubation at RT, the reaction was stopped byadding 20-30 ml medium or PBS. The spleen cells were centrifuged andwashed twice with PBS.

Single-Cell Sorting of Antigen-Specific CD8+ T Cells after CD137Staining

For antigen-specific restimulation 2.5×10{circumflex over ( )}6/wellspleen cells from immunized A2/DR1 mice were seeded in a 24-well plateand pulsed with a pool of overlapping peptides encoding the antigen ofinterest or a control antigen. After 24 h incubation cells wereharvested, stained with a FITC-conjugated anti-CD3 antibody, aPE-conjugated anti-CD4 antibody, a PerCP-Cy5.5-conjugated anti-CD8antibody and a Dylight-649-conjugated anti-CD137 antibody. Sorting wasconducted on a BD FACS Aria flow cytometer (BD Biosciences). Cellspositive for CD137, CD3 and CD8 were sorted, one cell per well washarvested in a 96-well V-bottom-plate (Greiner Bio-One) containing humanCCD-1079Sk cells as feeder cells, centrifuged at 4° C. and storedimmediately at −80° C.

RNA Extraction, SMART-Based cDNA Synthesis and Unspecific Amplificationfrom Sorted Cells

RNA from sorted T cells was extracted with the RNeasy Micro Kit (Qiagen,Hilden, Germany) according to the instructions of the supplier. Amodified BD SMART protocol was used for cDNA synthesis: BD PowerScriptReverse Transcriptase (BD Clontech, Mountain View, Calif.) was combinedwith oligo(dT)-T-primer long for priming of the first-strand synthesisreaction and TS-short (Eurogentec S. A., Seraing, Belgium) introducingan oligo(riboG) sequence to allow for creation of an extended templateby the terminal transferase activity of the reverse transcriptase andfor template switch (Matz, M. et al. (1999) Nucleic Acids Res. 27,1558-1560). First strand cDNA synthesized according to themanufacturer's instructions was subjected to 21 cycles of amplificationwith 5 U PfuUltra Hotstart High-Fidelity DNA Polymerase (Stratagene, LaJolla, Calif.) and 0.48 μM primer TS-PCR primer in the presence of 200μM dNTP (cycling conditions: 2 min at 95° C. for, 30 s at 94° C., 30 sat 65° C., 1 min at 72° C. for, final extension of 6 min at 72° C.).Successful amplification of TCR genes was controlled with either humanor murine TCR-β constant region specific primers and consecutiveclonotype-specific human or murine Vα-/Vβ-PCRs were only performed ifstrong bands were detected.

First strand cDNA for the amplification of HLA class I or II sequenceswas synthesized with SuperScriptII Reverse Transcriptase (Invitrogen)and Oligo(dT) primer with 1-5 μg RNA extracted from patient-derivedPBMCs.

Design of PCR Primers for TCR and HLA Amplification

For design of human TCR consensus primers, all 67 TCR-Vβ and 54 TCR-Vαgenes (open reading frames and pseudogenes) as listed in theImMunoGeneTics (IMGT) database (http://www.imgt.org) together with theircorresponding leader sequences were aligned with the BioEdit SequenceAlignment Editor (e.g. http://www.bio-soft.net). Forward primers of 24to 27 bp length with a maximum of 3 degenerated bases, a GC-contentbetween 40-60% and a G or C at the 3′end were designed to anneal to asmany leader sequences as possible and equipped with a 15 bp 5′extensionfeaturing a rare restriction enzyme site and Kozak sequence. Reverseprimers were designed to anneal to the first exons of the constantregion genes, with primer TRACex1_as binding to sequences correspondingto amino acids 7 to 16 of Cα and TRBCex1_as to amino acids (aa) 8 to 16in Cβ1 and Cβ2. Both oligonucleotides were synthesized with a 5′phosphate. Primers were bundled in pools of 2-5 forward oligos withidentical annealing temperature.

This strategy was replicated for the design of murine TCR consensusprimers, aligning 129 listed TCR-Vα and 35 listed TCR-Vβ genes. Reverseprimers mTRACex1_as and mTRBCex1_as are homologous to sequencescorresponding to aa 24 to 31 and 8 to 15, respectively.

HLA consensus primers were designed by aligning all HLA class I and IIsequences listed on the Anthony Nolan Research Institute website(www.anthonynolan.com) with the BioEdit Sequence Alignment Editor.Forward primers of 23 to 27 bp length with a maximum of 3 degeneratedbut code-preserving bases annealing to as many as possible HLA sequencesof one locus were equipped with a 5′-phosphate and Kozak sequenceextension. Reverse primers were designed analogously but withoutintroduction of wobble bases and equipped with a 14 bp 5′-extensionencoding an AsiSI restriction enzyme site.

PCR Amplification and Cloning of V(D)J Sequences

3-6 μl of preamplified cDNA from isolated T cells was subjected to 40cycles of PCR in the presence of 0.6 μM Vα-/Vβ-specific oligo pool, 0.6μM Cα- or Cβ-oligo, 200 μM dNTP and 5 U Pfu polymerase (cyclingconditions: 2 min at 95° C., 30 s at 94° C., 30 s annealing temperature,1 min at 72° C., final extension time of 6 min at 72° C.). PCR productswere analyzed using Qiagen's capillary electrophoresis system. Sampleswith bands at 400-500 bp were size fractioned on agarose gels, the bandsexcised and purified using a Gel Extraction Kit (Qiagen, Hilden,Germany). Sequence analysis was performed to reveal the sequence of boththe V(D)J domain and 13 constant region, as TRBCex1_as and mTRBCex1_asprimer, respectively, match to both TCR constant region genes β1 and β2in human and mouse, respectively. DNA was digested and cloned into theIVT vectors containing the appropriate backbone for a complete TCR-α/βchain.

Flow Cytometric Analyses

Cell surface expression of transfected TCR genes was analyzed by flowcytometry using PE-conjugated anti-TCR antibody against the appropriatevariable region family or the constant region of the TCR β chain(Beckman Coulter Inc., Fullerton, USA) and FITC-/APC-labeledanti-CD8/-CD4 antibodies (BD Biosciences). Cell surface expression oftransfected CARs was analyzed using a Dylight-650-conjugatedidiotype-specific antibody (Ganymed Pharmaceuticals) recognizing thescFv fragment contained in all CLDN6-CAR constructs. HLA antigens weredetected by staining with FITC-labeled HLA class II-specific (BeckmanCoulter Inc., Fullerton, USA) and PE-labeled HLA class I-specificantibodies (BD Biosciences). CLDN6 surface expression on target cellswas analyzed by staining with an Alexa-Fluor647-conjugatedCLDN6-specific antibody (Ganymed Pharmaceuticals). Flow cytometricanalysis was performed on a FACS CANTO II flow cytometer using the FACSDiva software (BD Biosciences).

Luciferase Cytotoxicity Assay

For assessment of cell-mediated cytotoxicity a bioluminescence-basedassay was established as an alternative and optimization to ⁵¹Crrelease. In contrast to the standard chromium release assay, this assaymeasures lytic activity of effector cells by calculating the number ofviable luciferase expressing target cells following coincubation. Thetarget cells were stably or transiently transfected with the luciferasegene coding for the firefly luciferase from firefly Photinus pyralis (EC1.13.12.7). Luciferase is an enzyme catalyzing the oxidation ofluciferin. The reaction is ATP-dependent and takes place in two steps:

luciferin+ATP→luciferyl adenylate+PP_(i)

luciferyl adenylate+O₂→oxyluciferin+AMP+light

Target cells were plated at a concentration of 10⁴ cells per well inwhite 96-well plates (Nunc, Wiesbaden, Germany) and were cocultivatedwith varying numbers of TCR-transfected T cells in a final volume of 100μl. 3 h later 50 μl of a D-Luciferin (BD Biosciences) containingreaction mix (Luciferin (1 μg/μl), HEPES-buffer (50 mM, pH), Adenosine5′-triphosphatase (ATPase, 0.4 mU/μl, Sigma-Aldrich, St. Louis, USA) wasadded to the cells. By addition of ATPase to the reaction mixluminescence resulting from luciferase released from dead cells wasdiminished.

After a total incubation time of 4 h bioluminescence emitted by viablecells was measured using the Tecan Infinite 200 reader (Tecan,Crailsheim, Germany) Cell-killing activity was calculated in regard toluminescence values obtained after complete cell lysis induced by theaddition of 2% Triton-X 100 and in relationship to luminescence emittedby target cells alone. Data output was in counts per second (CPS) andpercent specific lysis was calculated as follows:

(1−(CPS_(exp)−CPS_(min))/(CPS_(max)−CPS_(min))))*100.

Maximum luminescence (maximum counts per second, CPSmax) was assessedafter incubating target cells without effectors and minimalluminescences (CPSmin) was assessed after treatment of targets withdetergent Triton-X-100 for complete lysis.

ELISPOT (Enzyme-Linked ImmunoSPOT Assay)

Microtiter plates (Millipore, Bedford, Mass., USA) were coated overnightat room temperature with an anti-IFNγ antibody 1-D1k (Mabtech,Stockholm, Sweden) and blocked with 2% human albumin (CSL Behring,Marburg, Germany) 2-5×10⁴/well antigen presenting stimulator cells wereplated in triplicates together with 0.3-3×10⁵/well TCR-transfected CD4+or CD8+ effector cells 24 h after electroporation. The plates wereincubated overnight (37° C., 5% CO₂), washed with PBS 0.05% Tween 20,and incubated for 2 hours with the anti-IFNγ biotinylated mAB 7-B6-1(Mabtech) at a final concentration of 1 μg/ml at 37° C. Avidin-boundhorseradish peroxidase H (Vectastain Elite Kit; Vector Laboratories,Burlingame, USA) was added to the wells, incubated for 1 hour at roomtemperature and developed with 3-amino-9-ethyl carbazole (Sigma,Deisenhofen, Germany)

CFSE (Carboxyfluorescein Succinimidyl Ester) Proliferation Assay

CD8+ T cells were transfected with TCR or CAR RNA and labeled with 2.5μM CFSE. Labeled T cells were washed and cocultured with RNA-transfectedautologous monocytes or iDCs (E:T (effector cells: target(tumor)cells)=10:1). After 4 days of coculture cells were harvested andproliferation was analyzed by flow cytometry based on the progressivehalving of CFSE fluorescence within daughter cells following celldivisions.

Retroviral Construct for Stable CAR Expression

For stable expression of the CLDN6-CAR or the CAR against an unrelatedtumor antigen used as a negative control the retroviral SIN vectorES12.6 was used (FIG. 19).

Transduction of Human T Cells

For the mouse adoptive cell transfer (ACT) experiments, human Tlymphocytes were enriched from PBMCs of healthy donors by removal ofmonocytes after 2 h of plastic adherence. T lymphocytes were cultured inX-Vivo15 (Lonza) medium supplemented with 5% human AB serum(Invitrogen), 100 U/ml IL2 (Proleukin S, Novartis), 20 ng/ml IL7(Miltenyi), 10 ng/ml IL15 (Miltenyi) and stimulated with magneticanti-CD3/anti-CD28 beads (Dynabeads; Invitrogen) at a 1:3 CD3 cell tobead ratio and transduced on days 3 and 4 post stimulation withretroviral supernatants. Cells were expanded in X-Vivo15 mediumsupplemented with 5% human AB serum, 300 U/ml IL2, 20 ng/ml IL7 and 10ng/ml IL15. Incubation 37° C., 5% CO₂, 95% rH (FIG. 20).

Mouse Model for In-Vivo Validation of Antitumoral Activity

Xenograft tumors were established by subcutaneous injection of 1×10⁷OV90-SC12 human ovarian tumor cells into 8-14 week-old NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice (The Jackson Laboratory, Bar Harbor, Me.).After 4 days, mice were treated with a single intravenous injection of1×10⁷ of CAR transduced T cells (20-37% CAR positive). Tumor monitoringwas performed weekly by volume measurements using caliper (FIG. 21(a)).

Example 2: Isolation of High-Affinity HLA-A*02-Restricted Murine TCRsSpecific for Claudin-6

We validated the immunogenic potential of CLDN6 in A2/DR1 mice byrepetitive intranodal immunization with CLDN6 encoding IVT-RNA and usedspleen cells of these mice for isolation of CLDN6-specific T cells andsubsequent cloning of the corresponding TCR genes (FIG. 5). Spleen cellsof immunized mice were analyzed for the successful induction ofCLDN6-specific T cells and their reactivity against predicted HLA-A*02binding CLDN6 peptides ex-vivo by IFNγ-ELISPOT assay (FIG. 6).

Significant frequencies of CLDN6-specific T cells could be induced Inall three mice by RNA immunization, whereas T cell reactivity wasfocused on two CLDN6 peptides predicted, that were with the bestHLA-A*02 binding score (Cl6-A2-1 and Cl6-A2-2).

For isolation of CLDN6-specific T cells, spleen cells of immunized micewere restimulated in-vitro and sorted by flow cytometry based on theactivation-induced upregulation of CD137 (FIG. 7).

CLDN6-specific CD8+ T cells could be retrieved from all three immunizedA2/DR1 mice and a total of 11 CLDN6-specific TCRs were cloned fromsingle-sorted murine T cells.

TCRs were subjected to immunological validation assays, which revealedthat six CLDN6-TCRs recognized the HLA-A*0201-restricted epitopeCLDN6-91-99 (Cl6-A2-1) and four CLDN6-TCRs were specific for CLDN6-14-22(Cl6-A2-2), whereas both epitopes were previously identified by ex-vivoELISPOT analysis (FIG. 8). One CLDN6-TCR (TCR_(CD8)-CLDN6#7) recognizedthe peptide CLDN6-7-15 (Cl6-A2-3).

Example 3: Comparative Testing of Murine TCRs Specific for CLDN6 91-99

In total six murine TCRs were identified that all recognize theHLA-A*02-restricted epitope CLDN6-91-99. In order to confirm that thisepitope is naturally processed and presented by endogenously CLDN6expressing tumor cell lines and to evaluate the potential of theidentified murine TCRs to mediate killing of such cells aluciferase-based cytotoxicity assay was performed. Human preactivatedCD8+ T cells were transfected with TCR RNA and surface expression wasanalyzed by flow cytometry (FIG. 9). All murine TCRs were expressed on ahigh percentage of human CD8+ T cells after RNA transfer as indicated bystaining with an fluorochrome-conjugated antibody specific for theconstant domain of the murine TCR-β chain. TCR-transfected T cells weresubjected to luciferase-based cytotoxicity assay together with theCLDN6-expressing tumor cell lines PA1 (teratoma) and NIH-Ovcar3 (ovariancarcinoma). The CLND6-negative breast cancer cell line MDA-MB-231 servedas negative control. All TCRs mediated efficient lysis ofCLDN6-expressing tumor cell lines ranging from 38-94% of PA1 and 29-76%of NIH-Ovcar3, while no lysis could be observed with untransfected Tcells (FIG. 10). Most target cells were lysed when the mTCR_(CD8)-CLDN6#1, #8 or #18 were used. In order to analyze, if the murine TCRs canmediate specific proliferation of human T cells after coculture withautologous antigen-expressing target cells a CFSE proliferation assaywas performed (FIG. 11). TCR-transfected CD8+ T cells were coculturedwith autologous monocytes transfected with titrated amounts of CLDN6RNA. All TCRs mediated specific proliferation indicated by the dilutionof the CFSE proliferation dye after 4 days of coculture withCLDN6-RNA-transfected CD14+ cells, whereas again mTCR_(CD8)-CLDN6 #1, #8or #18 showed the best results, especially when low amounts of CLDN6 RNAwere transfected into the target cells. We decided to usemTCR_(CD8)-CLDN6 #18 as a gold standard for the lead structure selectiontogether with CLDN6 targeting CAR formats.

Example 4: Generation and In-Vitro Validation of Claudin-6-Specific CARs

We evaluated two different CAR formats to specifically target CLDN6 onCLDN6 expressing target cells. One of them represents a novel formatbased on the linkage of the scFv with the constant domain of the murineTCRβ chain and coexpression of the constant domain of the TCRα chain(CAR/Cα) (Voss R H et al., (2011) Molecular Therapy 19, supplement, S86)(FIG. 3). The second format represents a classical 2nd generation CAR(CAR-28ζ) that contains the signaling and costimulatory moieties of CD3ζand CD28, respectively. A deletion of the lck binding moiety in the CD28endodomain abrogates IL2 secretion upon CARengagement to preventinduction of regulatory T cells (Kofler D. M. et al., (2011) MolecularTherapy 19 (4), 760-767). A modification of the IgG1 Fc ‘spacer’ domainin the extracellular moiety of the CAR avoids ‘off-target’ activationand unintended initiation of an innate immune response (Hombach A. etal., (2010) Gene Therapy 17, 1206-1213).

As CARs provide HLA independent scFv-mediated antigen-binding they arefunctional in both CD4+ and CD8+ T cells. Therefore, we first analyzedthe CAR surface expression on CD4+ and CD8+ T cells after transfectionof CAR RNA into bulk PBMCs.

Both, the novel CAR/Cα and the classical 2nd generation CAR (CAR-28ζ)are expressed on the surface of human T cells after RNA transfer (FIG.12). The CAR-2ζ was significantly better expressed on the surface ofCD4+ and CD8+ T cells than the CAR/Cα. The latter one was transferredeither by cotransfection of the CAR and the Cα chain or as a 2Apeptide-based bicistronic vector for simultaneous expression of CAR andCα genes. Flow cytometry analysis demonstrated that the 2A-based linkageof CAR and Cα results in decreased surface expression compared tocoexpression of the two components. As a bicistronic vector would beused for clinical testing the linkage of the two CAR/Cα components hasto be further improved.

To analyze the specific tumor cell lysis mediated by the differentCLDN6-targeting receptor formats a luciferase-based cytotoxicity assaywas performed. CAR- or TCR-transfected preactivated CD8+ T cells werecultured with CLDN6-positive or negative tumor cell lines at differenteffector-to-target ratios and the specific lysis was calculated after 4h of coculture (FIG. 13). All CAR- and TCR-transfected T cellsdemonstrated significant specific lysis of CLDN6 expressing tumor celllines compared to untransfected T cells.

A prerequisite for the proliferation and persistence of CAR-engineered Tcells in the patient is the presence of antigen as demonstrated bypromising clinical trial results of CD19-specific CARs in hematologicmalignancies. In analogy to the expansion of endogenous T cells by RNAimmunization, we wanted to analyze, if CAR T cells could also beexpanded using RNA-vaccination of target cells to provide natural CLND6for CAR T cell stimulation. An in vitro proliferation assay wasperformed using CAR-transfected CD8+ T cells together with CLDN6 orcontrol RNA-transfected autologous iDCs (FIG. 14). The mTCR_(CD8)-CLDN6#18 mediated best proliferation in response to CLDN6-transfected targetcells (73%). The CLDN6-CAR-28ζ also resulted in a significant proportionof proliferating T cells (44%), while the CLDN6-CAR/Cα failed to induceproliferation probably due to the lack of CD28-mediated costimulation.As induction of proliferation is a prerequisite for successfulantitumoral activity, we decided to use CAR-28ζ format for further leadstructure selection.

Example 5: CLDN6-CAR-28ζ Lead Structure Selection for Preclinical andClinical Testing

The CLDN6-CAR-2ζ scFv fragment that is responsible for antigenrecognition contains an unpaired cysteine. As such a free cysteine couldresult in misfolding of the CAR protein under certain circumstances orin unwanted interactions with other cyteines by the formation ofdisulfide bonds, we decided to eliminate this cysteine and exchanged itby a serine, a glycine or an alanine.

We than comparatively analyzed the resulting CLDN6-CAR-2ζ constructsregarding surface expression (FIG. 15, 16) and cytotoxicity (FIG. 17).Except of the glycine variant all mutated constructs demonstratedsurface expression and lysis comparable to the wild-type variant.

In order to compare the affinity of the mutated CAR constructs theircytotoxic potential in response to autologous iDCs transfected withtitrated amounts of CLDN6 RNA was analyzed. Even extremely littleamounts of CLDN6 RNA (0.001 μg) resulted in significant lysis of targetcells mediated by all CAR constructs. As the serine variant of theCLDN6-CAR-28ζ showed slightly better results regarding surfaceexpression and cytotoxicity, we decided to use this variant forpreclinical testing.

Example 6: In-Vivo Antitumoral Activity of the CLDN6-CAR

After having determined the antitumor activity against CLDN6 expressingtumor cell lines in-vitro the antitumor ability in tumor-bearing micewas determined. Therefore, the potency of CLDN6-CAR transduced human Tcells was compared to T cells transduced with a control CAR against anunrelated tumor antigen and untransduced T cells in a xenograft model. Atotal of 1×10⁷ cells of the human ovarian carcinoma cell line OV90-SC12were injected subcutaneously in NSG mice. Four days after tumorengraftment the mice were treated with a single intravenous injection of1×10⁷ of CAR-transduced T cells. Tumor monitoring was performed weeklyby volume measurements using caliper. Treatment of the mice withCLDN6-CAR-transduced T cells significantly slowed tumor growth comparedto control groups treated with unrelated tumor antigen-CAR-transduced,untransduced T cells or a group not receiving T cells (FIGS. 21 (b) and(c)).

Example 7: In-Vitro Proliferation of CLDN6-CAR T Cells in Response toCLDN6-Expressing Target Cells

In analogy to the expansion of endogenous T cells by RNA immunization,the stimulation and expansion of CAR T cells using RNA-vaccination oftarget cells to provide natural CLND6 was analyzed by in vitroproliferation assay. CD8⁺ T cells were transfected with IVT-RNA encodinga CAR against CLDN6 or an unrelated tumor antigen as negative control,labeled with CFSE (carboxyfluorescein succinimidyl ester) and coculturedwith CLDN6-transfected autologous iDCs for 4 days (FIG. 22). TheCLDN6-CAR mediated proliferation of nearly all CD8⁺ T cells in responseto CLDN6-transfected iDC could be observed (95%), while only backgroundproliferation (1.5%) could be observed for unrelated tumor antigen-CARtransfected T cells indicating that proliferation was not depending onthe CAR backbone but was CLDN6-specific.

CLDN6-specific T cell epitopes A2-1 (aa 91-99) ALFGLLVYL A2-2 (aa 14-22)TLLGWVNGL A2-3 (7-15) QILGVVLTL CLDN6-specific T cell receptorsTCR_(CD8)-mC16#1: SEQ ID NO: 6; > Vα9N.3 J13 CMLLALLSVLGIHFLLRDAQAQSVTQPDARVTVSEGASLQLRCKYSYFGTPYLFWYVQYPRQGLQLLLKYYPGDPVVQGVNGFEAEFSKSNSSFHLRKASVHWSDWAVYFCAVSMSSGTYQRFGTGTKLQVVPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 7; > Vβ29 D1 J2.5 C2MRVRLISAVVLCFLGTGLVDMKVTQMPRYLIKRMGENVLLECGQDMSHETMYWYRQDPGLGLQLIYISYDVDSNSEGDIPKGYRVSRKKREHFSLILDSAKTNQTSVYFCASSSQNQDTQYFGPGTRLLVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGL VLMAMVKKKNSTCR_(CD8)-mC16#2: SEQ ID NO: 8; > Vα6N.6 J23 CMDSFPGFVAVILLILGRTHGDSVTQTEGQVTVSESKSLIINCTYSATSIGYPNLFWYVRYPGEGLQLLLKVITAGQKGSSRGFEATYNKEATSFHLQKASVQESDSAVYYCALNNQGKLIFGQGTKLSIKPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 9; > Vβ13.2 D1 J2.4 C2MGSRLFFVLSSLLCSKHMEAAVTQSPRNKVAVTGGKVTLSCNQTNNHNNMYWYRQDTGHGLRLIHYSYGAGSTEKGDIPDGYKASRPSQENFSLILELATPSQTSVYFCASGGDSQNTLYFGAGTRLSVLEDLRNVTPPKVSLFE,PSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVL MAMVKKKNSTCR_(CD8)-mC16#3: SEQ ID NO: 18; > Vα16N J6 CMLILSLLGAAFGSICFAATSMAQKVTQTQTSISVVEKTTVTMDCVYETRDSSYFLFWYKQTASGEIVFLIRQDSYKKENATVGHYSLNFQKPKSSIGLIITATQIEDSAVYFCAMRDSSGGNYKPTFGKGTSLVVHPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 19; > Vβ2 D2 J2.4 C2MGSIFLSCLAVCLLVAGPVDPKIIQKPKYLVAVTGSEKILICEQYLGHNAMYWYRQSAKKPLEFMFSYSYQKLMDNQTASSRFQPQSSKKNHLDLQITALKPDDSATYFCASSQEDWGSQNTLYFGAGTRLSVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS TCR_(CD8)-mC16#7:SEQ ID NO: 28; > Vα6N.7 or Vα6D.7_4 J26 CMDSFPGFMTVMLLIFTRAHGDSVTQTEGQVALSEEDFLTIHCNYSASGYPALFWYVQYPGEGPQFLFRASRDKEKGSSRGFEATYDKGTTSFHLRKASVQESDSAVYYCALGNNYAQGLTFGLGTRVSVFPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 29; > Vβ13.3 D1 J1.4_02 C1MGSRLFFVVLILLCAKHMEAAVTQSPRSKVAVTGGKVTLSCHQTNNHDYMYWYRQDTGHGLRLIHYSYVADSTEKGDIPDGYKASRPSQENFSLILELASLSQTAVYFCASSTGNERLFFGHGTKLSVLEDLRNVTPPKVSLl-E,PSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVM AMVKRKNSTCR_(CD8)-mC16#8: SEQ ID NO: 10; > Vα16N J13 CMLILSLLGAAFGSICFATSMAQKVTQTQTSISVVEKTTVTMDCVYETRDSSYFLFWYKQTASGEIVFLIRQDSYKKENATVGHYSLNFQKPKSSIGLIITATQIEDSAVYFCAMREAANSGTYQRFGTGTKLQVVPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 11; > Vβ2 D1 J1.3 C1MGSIFLSCLAVCLLVAGPVDPKIIQKPKYLVAVTGSEKILICEQYLGHNAMYWYRQSAKKPLEFMFSYSYQKLMDNQTASSRFQPQSSKKNHLDLQITALKPDDSATYFCASSQQNSGNTLYFGEGSRLIVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLV VMAMVKRKNSTCR_(CD8)-mC16#10: SEQ ID NO: 20; > Vα13D.4_03 J42 CMKRLVCSLLGLLCTQVCWVKGQQVQQSPASLVLQEGENAELQCNFSSTATRLQWFYQRPGGSLVSLLYNPSGTKHTGRLTSTTVTKERRSSLHISSSQTTDSGTYFCAMSSNSGGSNAKLTFGKGTKLSVKSNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 21; > Vβ4_02 D2 J2.7 C2MGCRLLSCVAFCLLGIGPLETAVFQTPNYRVTRVGNEVSFNCEQTLDHNTMYWYKQDSKKLLKIMFSYNNKQLIVNETVPRRFSPQSSDKAHLNLRIKSVELEDSAVYLCASSDWGDSYEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGL VLMAMVKKKNSTCR_(CD8)-mC16#12: SEQ ID NO: 12; > Vα3.3 J50 CMKTVTGPLFLCFWLQLNCVSRGEQVEQRPPHLSVREGDSAVITCTYTDPNSYYFFWYKQEPGASLQLLMKVFSSTEINEGQGFTVLLNKKDKRLSLNLTAAHPGDSAAYFCAVESSSFSKLVFGQGTSLSVVPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 13; > Vβ26 D2 J2.5 C2MATRLLCYTVLCLLGARILNSKVIQTPRYLVKGQGQKAKMRCIPEKGHPVVFWYQQNKNNEFKFLINFQNQEVLQQIDMTEKRFSAECPSNSPCSLEIQSSEAGDSALYLCASSLTGGAQDTQYFGPGTRLLVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS TCR_(CD8)-mC16#13: SEQ ID NO: 22; > Vα16N J22 CMLILSLLGAAFGSICFAATSMAQKVTQTQTSISVVEKTTVTMDCVYETRDSSYFLFWYKQTASGEIVFLIRQDSYKKENATVGHYSLNFQKPKSSIGLIITATQIEDSAVYFCAMRVASSGSWQLIFGSGTQLTVMPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 23; > Vβ2 D1 J2.1 C2MGSIFLSCLAVCLLVAGPVDPKIIQKPKYLVAVTGSEKILICEQYLGHNAMYWYRQSAKKPLEFMFSYSYQKLMDNQTASSRFQPQSSKKNHLDLQITALKPDDSATYFCASSQGDNNYAEQFFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS TCR_(CD8)-mC16#14:SEQ ID NO: 14; > Vα4N.4 or Vα4D.4_03 J6 CMQRNLVAVLGILWVQICWVRGDQVEQSPSALSLHEGTGSALRCNFTTTMRAVQWFRKNSRGSLINLFYLASGTKENGRLKSAFDSKERYSTLHIRDAQLEDSGTYFCAAEGGGNYKPTFGKGTSLVVHPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 15; > Vβ31 D1 J1.1 C1MLYSLLAFLLGMFLGVSAQTIHQWPVAEIKAVGSPLSLGCTIKGKSSPNLYWYWQATGGTLQQLFYSITVGQVESVVQLNLSASRPKDDQFILSTEKLLLSHSGFYLCAWSPPINTEVFFGKGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVM AMVKRKNSTCR_(CD8)-mC16#15: SEQ ID NO: 24; > Vα3.1 J39 CMKTVTGPLLLCFWLQLNCVSRGEQVEQRPPHLSVREGDSAIIICTYTDSATAYFSWYKQEAGAGLQLLMSVLSNVDRKEEQGLTVLLNKKDKRLSLNLTAAHPGDSAVYFCATNAGAKLTFGGGTRLTVRPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 25; > Vβ4 D2 J2.7 C2MGCRLLSCVAFCLLGIGPLETAVFQTPNYHVTQVGNEVSFNCKQTLGHDTMYWYKQDSKKLLKIMFSYNNKQLIVNETVPRRFSPQSSDKAHLNLRIKSVEPEDSAVYLCASSLYWGDSYEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS TCR_(CD8)-mC16#17:SEQ ID NO: 26; > Vα14.3 or Vα14D.3/DV8_08 J22 CMDKNLTASFLLLGLHLAGVSGQQEKRDQQQVRQSPQSLTVWEGETAILNCSYENSAFDYFPWYQQFPGEGPALLISILSVSDKKEDGRFTIFFNKREKKLSLHIADSQPGDSATYFCAASLSSGSWQLIFGSGTQLTVMPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 27; > Vβ3 D2 J2.7 C2MDIWLLGWIIFSFLEAGHTGPKVLQIPSHQIIDMGQMVTLNCDPVSNHLYFYWYKQILGQQMEFLVNFYNGKVMEKSKLFKDQFSVERPDGSYFTLKIQPTALEDSAVYFCASSLVGGYEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGL VLMAMVKKKNSTCR_(CD8)-mC16#18: SEQ ID NO: 16; > Vα6D.6_02 J4 CMDSSPGFVAVILLILGRTHGDSVTQTEGPVTVSESESLIINCTYSATSIAYPNLFWYVRYPGEGLQLLLKVITAGQKGSSRGFEATYNKETTSFHLQKASVQESDSAVYYCALGETGSFNKLTFGAGTRLAVCPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 17; > Vβ26 D1 J2.7 C2MATRLLCYTVLCLLGARILNSKVIQTPRYLVKGQGQKAKMRCIPEKGHPVVFWYQQNKNNEFKFLINFQNQEVLQQIDMTEKRFSAECPSNSPCSLEIQSSEAGDSALYLCASSLGIYEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLM AMVKKKNS

1-43. (canceled)
 44. An artificial T cell receptor comprising: a bindingdomain for claudin-6 (CLDN6); a costimulatory domain selected from thegroup consisting of: CD28, CD137 (4-1BB), CD134 (OX40), and CD278(ICOS); and a T cell signaling domain that activates cytotoxiclymphocytes upon binding to CLDN6.
 45. The artificial T cell receptor ofclaim 44, wherein the costimulatory domain is CD137 (4-1BB).
 46. Theartificial T cell receptor of claim 44, further comprising atransmembrane domain.
 47. The artificial T cell receptor of claim 46,further comprising a spacer region which links the binding domain forCLDN6 to the transmembrane domain.
 48. The artificial T cell receptor ofclaim 44, wherein the T cell signaling domain comprises the endodomainof CD3-zeta or a functional fragment thereof.
 49. The artificial T cellreceptor of claim 44, wherein the binding domain for CLDN6 comprises thecomplementary-determining regions CDR1, CDR2 and CDR3 of the heavy chainvariable region (VH) according to SEQ ID NO: 32 and thecomplementary-determining regions CDR1, CDR2 and CDR3 of the light chainvariable region (VL) according to SEQ ID NO:
 39. 50. The artificial Tcell receptor of claim 44, wherein said heavy chain variable region (VH)and the corresponding light chain variable region (VL) are connected viaa peptide linker comprising the amino acid sequence (GGGGS)3.
 51. Theartificial T cell receptor of claim 44, wherein the binding domain forCLDN6 comprises a VH comprising an amino acid sequence represented bySEQ ID NO:
 32. 52. The artificial T cell receptor of claim 44, whereinthe binding domain for CLDN6 comprises a VL comprising an amino acidsequence represented by SEQ ID NO:
 39. 53. The artificial T cellreceptor of claim 44, wherein the binding domain for CLDN6 comprises aVH comprising an amino acid sequence represented by SEQ ID NO: 32 and aVL comprising an amino acid sequence represented by SEQ ID NO:
 39. 54.The artificial T cell receptor of claim 44, wherein the binding domainfor CLDN6 comprises an amino acid sequence represented by SEQ ID NO: 40.55. The artificial T cell receptor of claim 44 which comprises a signalpeptide which directs the nascent protein into the endoplasmicreticulum.